Abstract Study question Is double blastocysts transfer (DET) better than sequential single blastocyst transfer (seq-SET) in freeze all cycles? Summary answer Sequential single blastocyst transfer provides a higher live birth rate (LBR) per cycle initiated and eliminates multiple births in freeze-all cycles. What is known already Improvements in cryopreservation technology helped freeze-all strategy gain much popularity. The new debate is whether guidance for single embryo transfer should also be applied to frozen-thawed embryo transfers in freeze-all cycles. Study design, size, duration We performed a retrospective cohort analysis of 860 women in whom the entire cohort of embryos frozen at the blastocyst stage for various indications. All women aged 19–43 years, who had at least two blastocysts frozen and subsequently thawed and transferred were included. Preimplantation genetic testing cycles were excluded.The study period ranged from January 2016 to May 2019. Participants/materials, setting, methods Data regarding female age, number of embryos transferred, multiple pregnancy and live birth rates (LBR) were extracted from the electronic database. Women were categorized based on their age and the mode of embryo transfer. Primary outcome was live birth rate LBR per cycle initiated. Secondary outcomes were LBR per embryo transfer and multiple birth rate. Groups were compared using Fisher’s test, generalized estimating equation model and logistic regression analysis to adjust for confounding factors. Main results and the role of chance The study group comprised of 666 women (371 Seq-SET and 295 DET) who underwent 837 embryo transfer cycles. Second embryo transfer was affected in 46.1% of women in the Seq-SET group. Age, indication for freeze-all, and mode of transfer were related with the LBR. For women ≤ 35 (n = 370), LBRs per embryo transfer were similar in single and double embryo transfers (53.9% versus 64.2% respectively, p = 0.006, aOR=0.65, 95% CI:0.41–1.01). However, LBR per cycle initiated was significantly higher in Seq-SET group (78.9% versus 64.2% respectively, p = 0.004, aHR=2.09, 95% CI:1.28–3.41). While only one monochorionic twin delivery was observed with Seq-SET (0.5%), 19 out of 70 (27.1%) live births after DET were twins. For women >35 of age (n = 296) the likelihood of a live birth per embryo transfer was lower in single compared to double embryo transfers (33.2% versus 46.2%, respectively, p = 0.012, aOR=0.58 95% CI:0.38–0.88). Although LBR per cycle initiated was higher in Seq-SET (58.2%) than DET (46.2%), the difference did not reach statistical significance (p = 0.054, aHR=1.62, 95% CI:1.00–2.60). While no twin delivery was observed with Seq-SET, 8 out of 86 (9.3%) live births with DET were twins. Limitations, reasons for caution This was a retrospective study with small sample size performed at a single fertility center, which may limit the generalizability of our findings. Cost-efficiency was not studied. Wider implications of the findings: Seq-SET is associated with a comparable or higher likelihood of live birth per cycle initiated and a very low risk of twins when compared to DET. However, half of SET cases had to undergo two transfer cycles. Trial registration number NA
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