The efficacy of sequential day 3 embryo and blastocyst transfer in patients with repeated implantation failure

Published:January 06, 2023DOI:



      To evaluate the efficacy of sequential transfer that one cleavage-stage embryo on day 3 and one blastocyst on day 5 are sequentially transferred in the same treatment cycle over conventional day 3 embryo transfer and blastocyst transfer in patients with repeated implantation failure (RIF).
      Study Design: 2836 frozen embryo transfer (FET) cycles in patients with RIF were divided into three groups according to female age: < 35, 35-39 and > 39 years old groups, and four groups depending on the number and period of embryo transferred: two day 3 embryo, two blastocyst, single blastocyst and sequential transfer groups; Pregnancy outcomes including implantation rate (IR), clinical pregnancy rate (CPR), abortion rate (AR), ectopic pregnancy rate (EPR), multiple pregnancy rate (MPR), live birth rate (LBR) and neonatal characteristics from all the groups were assessed.


      Sequential transfer caused a significant increase in the IR, CPR and LBR over two day 3 embryo transfer and did not improve the IR, CPR and LBR over two blastocyst transfer in patients with RIF. Sequential transfer had higher CPR, MPR and LBR and lower IR than single blastocyst transfer. No significant differences were present in neonatal characteristics among the transfer protocol groups. Singleton group had a higher average gestational age and birthweight as well as a lower cesarean section rate, preterm labor rate and low birthweight rate than twin group. Additionally, the AR had no significant difference and the EPR of blastocyst transfer was low.


      Sequential transfer was not an effective method to improve IR in patients with RIF, and blastocyst transfer with higher IR was suggested. Single blastocyst transfer could serve as an effective transfer protocol to reduce MPR.


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        • Mitri F.
        • Nayot D.
        • Casper R.F.
        • Bentov Y.
        Current tools for the optimization of embryo transfer technique for recurrent implantation failure.
        Minerva Ginecol. 2016; 68: 431-449
        • Sullivan E.A.
        • Wang Y.A.
        • Hayward I.
        • Chambers G.M.
        • Illingworth P.
        • McBain J.
        • et al.
        Single embryo transfer reduces the risk of perinatal mortality, a population study.
        Hum Reprod. 2012; 27: 3609-3615
        • Kaur P.
        • Swarankar M.L.
        • Maheshwari M.
        • Acharya V.
        A comparative study between cleavage stage embryo transfer at day 3 and blastocyst stage transfer at day 5 in in-vitro fertilization/intra-cytoplasmic sperm injection on clinical pregnancy rates.
        J Hum Reprod Sci. 2014; 7: 194-197
        • Fernández-Shaw S.
        • Cercas R.
        • Braña C.
        • Villas C.
        • Pons I.
        Ongoing and cumulative pregnancy rate after cleavage-stage versus blastocyst-stage embryo transfer using vitrification for cryopreservation: impact of age on the results.
        J Assist Reprod Genet. 2015; 32: 177-184
        • Glujovsky D.
        • Farquhar C.
        Cleavage-stage or blastocyst transfer: what are the benefits and harms?.
        Fertil Steril. 2016; 106: 244-250
        • Levitas E.
        • Lunenfeld E.
        • Har-Vardi I.
        • Albotiano S.
        • Sonin Y.
        • Hackmon-Ram R.
        • et al.
        Blastocyst-stage embryo transfer in patients who failed to conceive in three or more day 2–3 embryo transfer cycles: a prospective, randomized study.
        Fertil Steril. 2004; 81: 567-571
        • Bashiri A.
        • Halper K.I.
        • Orvieto R.
        Recurrent Implantation Failure-update overview on etiology, diagnosis, treatment and future directions.
        Reprod Biol Endocrinol. 2018; 16: 1-28
        • Fang C.
        • Huang R.
        • Li T.-T.
        • Jia L.
        • Li L.-L.
        • Liang X.-Y.
        Day-2 and day-3 sequential transfer improves pregnancy rate in patients with repeated IVF-embryo transfer failure: a retrospective case-control study.
        Reprod Biomed Online. 2013; 26: 30-35
        • Zhang J.
        • Wang C.
        • Zhang H.
        • Zhou Y.
        Sequential cleavage and blastocyst embryo transfer and IVF outcomes: a systematic review.
        Reprod Biol Endocrinol. 2021; 19: 1-12
        • Machtinger R.
        • Dor J.
        • Margolin M.
        • Levron J.
        • Baum M.
        • Ferber B.
        • et al.
        Sequential transfer of day 3 embryos and blastocysts after previous IVF failures despite adequate ovarian response.
        Reprod Biomed Online. 2006; 13: 376-379
        • Goto S.
        • Takebayashi K.
        • Shiotani M.
        • Fujiwara M.
        • Hirose M.
        • Noda Y.
        Effectiveness of 2-step (consecutive) embryo transfer. Comparison with cleavage-stage transfer.
        J Reprod Med. 2003; 48: 370-374
        • Torky H.
        • Ahmad A.
        • Hussein A.
        • El-Desouky E.S.
        • Aly R.
        • Ragab M.
        • et al.
        Comparing sequential vs day 3 vs day 5 embryo transfers in cases with recurrent implantation failure: randomized controlled trial.
        JBRA Assist Reprod. 2021; 25: 185-192
        • Almog B.
        • Levin I.
        • Wagman I.
        • Kapustiansky R.
        • Schwartz T.
        • Mey-Raz N.
        • et al.
        Interval double transfer improves treatment success in patients with repeated IVF/ET failures.
        J Assist Reprod Genet. 2008; 25: 353-357
        • Yazbeck C.
        • Jamaa N.B.
        • Hazout A.
        • Cohen-Bacrie P.
        • Junca A.-M.
        • Rougier N.
        Advantages of the two-step embryo transfer strategy in human IVF/ICSI cycles.
        Zygote. 2013; 21: 77-83
        • Kyono K.
        • Fukunaga N.
        • Chiba S.
        • Nakajo Y.
        • Fuchinoue K.
        • Yagi A.
        • et al.
        Two-step consecutive transfer of early embryos and blastocysts.
        Reprod Med Biol. 2003; 2: 133-137
        • Abramovici H.
        • Dirnfeld M.
        • Weisman Z.
        • Sorokin Y.
        • Lissak A.
        • Rofe A.
        • et al.
        Pregnancies following the interval double-transfer technique in an in vitro fertilization-embryo transfer program.
        J In Vitro Fert Embryo Transf. 1988; 5: 175-176
        • Gardner D.K.
        • Lane M.
        • Stevens J.
        • Schlenker T.
        • Schoolcraft W.B.
        Blastocyst score affects implantation and pregnancy outcome: towards a single blastocyst transfer.
        Fertil Steril. 2000; 73: 1155-1158
        • Ismail Madkour W.A.
        • Noah B.
        • Zaheer H.
        • Al-Bahr A.
        • Abdelhamid A.M.S.
        • Shaeer M.
        • et al.
        Does sequential embryo transfer improve pregnancy rate in patients with repeated implantation failure? A randomized control study.
        Middle East Fertility Society Journal. 2015; 20: 255-261
        • Hennes A.
        • Held K.
        • Boretto M.
        • De Clercq K.
        • Van den Eynde C.
        • Vanhie A.
        • et al.
        Functional expression of the mechanosensitive PIEZO1 channel in primary endometrial epithelial cells and endometrial organoids.
        Sci Rep. 2019; 9: 1-14
        • Lu X, Liu S, Dong X, Hospital Z, University F
        Research progress of two-step sequential embryo transfer strategy in in vitro fertilization-embryo transfer cycles.
        Chinese Journal of Reproduction and Contraception. 2018; 4: 333-336
        • Tehraninejad E.S.
        • Raisi E.
        • Ghaleh F.B.
        • Rashidi B.H.
        • Aziminekoo E.
        • Kalantari V.
        • et al.
        The sequential embryo transfer compared to blastocyst embryo transfer in in vitro fertilization (IVF) cycle in patients with the three repeated consecutive IVF.
        A randomized controlled trial. Gynecol Endocrinol. 2019; 35: 955-959
        • Stamenov G.S.
        • Parvanov D.A.
        • Chaushev T.A.
        Mixed double-embryo transfer: A promising approach for patients with repeated implantation failure.
        Clin Exp Reprod Med. 2017; 44: 105-110
        • Nadkarni P.
        • Nadkarni K.
        • Singh P.
        • Singh P.
        • Nadkarni A.
        • Kalyani K.
        • et al.
        A comparative study of pregnancy outcome of sequential versus day 3 versus only blastocyst (day 6) transfer at a single IVF center over one year.
        International Journal of Reproduction, Contraception, Obstetrics and Gynecology. 2015; : 2032-2035
        • Zhang B.
        • Cui L.
        • Tang R.
        • Ding L.
        • Yan L.
        • Chen Z.-J.
        • et al.
        Reduced Ectopic Pregnancy Rate on Day 5 Embryo Transfer Compared with Day 3: A Meta-Analysis.
        PLoS One. 2017; 12: e0169837
        • Fang C.
        • Huang R.
        • Wei L.-N.
        • Jia L.
        Frozen-thawed day 5 blastocyst transfer is associated with a lower risk of ectopic pregnancy than day 3 transfer and fresh transfer.
        Fertil Steril. 2015; 103: 655-661.e3
        • Sekhon L.
        • Lee J.
        • Mehta S.
        • Mukherjee T.
        • Sandler B.
        • Copperman A.B.
        Are Euploid Embryos Less Likely to Result in Ectopic Implantation? [16].
        Obstetrics & Gynecology. 2016; 127: 6S
        • Toikkanen S.
        • Joensuu H.
        • Erkkola R.
        DNA aneuploidy in ectopic pregnancy and spontaneous abortions.
        Eur J Obstet Gynecol Reprod Biol. 1993; 51: 9-13