Pregnancy and neonatal outcomes of sequential embryo transfer versus double cleavage-stage embryo transfer and double blastocyst transfer in frozen-thawed embryo transfer cycles: a retrospective cohort study

Sequential embryo transfer (Seq-ET), involving the transfer of a cleavage-stage embryo followed by a blastocyst within the same frozen-thawed embryo transfer (FET) cycle, has been proposed as an alternative strategy to improve reproductive outcomes. However, evidence regarding its effectiveness and neonatal safety compared to the traditional double embryo transfer (DET) in the general infertile population remains limited. This retrospective cohort study incorporated 2,959 FET cycles, using Seq-ET ( n = 165), and DET strategies at Day 3 cleavage (D3-DET, n = 1,043) or Day 5/6 blastocyst (D5/6-DET, n = 1,751) stage in our reproductive center (2023.07-2025.01). The Seq-ET group was separately matched with the D3-DET or D5/6-DET group by propensity score matching (PSM). Propensity score-adjusted logistic regression analyses were used to control for confounding factors. The live birth rate (LBR), biochemical pregnancy rate, clinical pregnancy rate (CPR), implantation rate, miscarriage rate, multiple pregnancy rate, multiple live birth rate and neonatal outcomes were analyzed between groups. Seq-ET had significantly higher biochemical pregnancy rate (81.99% vs. 63.52%, P < 0.001), CPR (67.08% vs. 43.57%, P < 0.001), and LBR (51.55% vs. 33.86%, P < 0.001) than D3-DET. Compared with D5/6-DET, Seq-ET showed comparable biochemical pregnancy rate, CPR, and LBR, but was associated with a significantly lower multiple pregnancy rate (20.91% vs. 33.94%, P = 0.014) and higher miscarriage rate (22.73% vs. 13.94%, P = 0.048). Neonatal outcomes were analyzed separately for singleton and twin live births. No significant differences were observed among singleton live births. Among twin live births, D5/6-DET had shorter gestational age at delivery, while other neonatal outcomes did not differ between groups. Seq-ET yielded superior pregnancy outcomes compared with D3-DET and achieved pregnancy outcomes comparable to D5/6-DET while reducing the risk of multiple pregnancy, although a higher miscarriage rate was observed in the Seq-ET group. Neonatal outcomes were generally comparable between groups, with the except of shorter gestational age at delivery for twin live births in D5/6-DET group. These findings indicate that Seq-ET may serve as a viable alternative double embryo transfer strategy in FET cycles.