Various factors affecting the outcomes and safety of assisted reproductive technology (ART) have gained increasing attention with the widespread application of this technique. Previous unsuccessful ART treatments may influence the outcomes of subsequent embryo transfer cycles, as reported in several observational studies. For example, an unsuccessful ART cycle has been associated with decreased odds of ongoing implantation [1]. McLernon et al. [2] developed a predictive model estimating the chances of live birth over multiple complete cycles using population data from 113,873 women. They found that the chance of live birth was 21% lower after two unsuccessful in vitro fertilization (IVF) treatments compared with one, and 56% lower after six unsuccessful IVF treatments than one unsuccessful IVF treatment. External validation of this model using updated UK data between January 2010 and December 2016 reached similar conclusions [3]. In addition, the number of previous failed treatments has been identified as a prognostic factor for poor outcomes in models predicting embryo transfer success [4]. Repeated treatments also increase patients’ economic burden and psychological stress. Thus, efforts should be made to reduce the likelihood of repeated failures before or during the next embryo transfer after an initial failure. In good-prognosis patients without risk factors related to the endometrium, metabolic or endocrinologic disorders, or immunological disease, embryo-related factors become the primary consideration after the first failed frozen-thawed embryo transfer [5]. Regarding embryo selection, unlike repeated implantation failure cases where clinicians often recommend preimplantation genetic screening for aneuploidy, in the second transfer cycle following an initial failure, both doctors and patients tend to focus on embryo developmental stage and number. Increasing the number of embryos transferred may improve the chances of pregnancy in subsequent treatments. In clinical practice, infertile couples often choose double embryo transfer after failure with single embryo transfer, in consultation with their physicians.

Elective single blastocyst transfer (eSBT) has been recommended in ART to improve outcomes while minimizing the risk of multiple pregnancy [6]. Several international professional organizations, including American Society for Reproductive Medicine (ASRM) and European Society of Human Reproduction and Embryology (ESHRE), have issued related guidelines [7, 8, 9]. Meta-analyses of individual patient data from randomized trials have shown that elective single embryo transfer is associated with a higher chance of term singleton live birth compared with double embryo transfer [10]. As blastocyst culture has become more common as a selection method, the adoption of eSBT has increased. However, extended culture carries the risk of having no available embryos if none develop into blastocysts. Although this outcome indicates low developmental potential, it results in loss of time and financial resources for patients. To mitigate this, embryologists often recommend freezing two high-quality cleavage-stage embryos as a backup in case blastocyst culture fails. This raises the question of which type of embryo should be transferred after failure of an initial eSBT.

ART has significantly contributed to the global increase in multiple pregnancies [11, 12, 13]. Although the rate of multiple births has reduced in recent years owing to single embryo transfer and advancements in IVF techniques [14], the twin pregnancy rate remains high, due to nonadherence to the guideline recommending elective single embryo transfer (eSET) [15]. Many patients who insist on multiple embryo transfer are unaware of the risks associated with multiple pregnancy, including preterm birth, low birth weight, preeclampsia, gestational diabetes, placental abruption, intrauterine growth restriction, and perinatal mortality [16, 17]. Furthermore, the risk of ectopic pregnancy increases with the number of embryos transferred, up to 20-fold [18]. Singleton pregnancies following double embryo transfer (DET) are also associated with higher risks of neonatal death and low birthweight compared with singleton pregnancies after SET in frozen embryo transfer cycles [19]. Additionally, twin pregnancies conceived through ART have higher risks than naturally conceived twin pregnancies [17]. Thus, reducing the number of embryos transferred in ART is essential.

In clinical practice, when an eSET fails, patients and clinicians often choose to transfer two cryopreserved embryos in the next cycle to increase the likelihood of success. However, it remains uncertain whether transferring two high-quality cleavage-stage embryos improves pregnancy outcomes compared with eSBT, and whether this strategy increases the risk of multiple pregnancy. Therefore, this study aimed to evaluate the outcomes of frozen-thawed embryo transfer (FET) cycles in patients who failed to conceive after an initial eSBT cycle and subsequently underwent either double vitrified-warmed high-quality cleavage embryo transfer (DC-ET) or eSBT, with a focus on comparing clinical pregnancy and multiple pregnancy rates.

Baseline characteristics of oocyte retrieval cycle are shown in Table 1. There were no significant differences in mean maternal or paternal age, body mass index (BMI), duration or type of infertility, basal follicle-stimulating hormone (FSH), LH, estrogen (E₂), antral follicle count, anti-Müllerian hormone (AMH), or parameters of controlled ovarian stimulation (COS) and fertilization protocols between the DC-ET and SBT groups. There were also no significant differences in the total number of oocytes, the number of mature oocytes available cleavage embryos, or endometrial thickness on the day of embryo transfer between the two groups. However, the number of available blastocysts was higher in the SBT group.

As the baseline characteristics of FET between DC-ET group and SBT group, the female age at the time of embryo transfer were slightly higher in SBT group, while the embryo thawing survival rates, the endometrium preparation protocols, the endometrial thickness on the day of embryo transferred, were similar between the two groups (Table 2).

We then compared pregnancy outcomes between the DC-ET and SBT groups (Table 3). The implantation rate was significantly higher in the SBT group than in the DC-ET group (48.94% vs. 30.74%, p 0.001, adjusted p = 0.002, odds ratio (OR): 2.023, 95% confidence interval (CI): 1.300–3.149). However, there were no significant differences in clinical pregnancy rate (48.94% vs. 51.64%, adjusted p = 0.083, OR: 0.550, 95% CI: 0.280–1.080), live birth rate (39.00% vs. 39.34%, adjusted p = 0.220, OR: 0.658, 95% CI: 0.337–1.284), or pregnancy loss rate (18.84% vs. 20.63%, adjusted p = 0.892, OR: 1.076, 95% CI: 0.373–3.106) between the two groups. In contrast, the multiple pregnancy rate was significantly lower in the SBT group (2.90% vs. 20.63%, adjusted p = 0.007, OR: 0.113, 95% CI: 0.023–0.549).

This retrospective study showed no significant differences in pregnancy outcomes between eSBT and double high-quality cleavage embryo transfer after failure of an elective single vitrified-warmed blastocyst transfer cycle. However, the multiple pregnancy rate was much lower after eSBT compared with double cleavage embryo transfer. Therefore, eSBT appears to be the optimal choice in patients who have experienced failure in previous FET cycles.

Several studies have compared pregnancy outcomes across cycles with different numbers of embryos transferred. A meta-analysis of eighty-five studies (14 randomized controlled trials and 71 observational studies) concluded that in women younger than 40 years or in the presence of at least one high-quality embryo, single embryo transfer should be incorporated into clinical practice [21]. Another study evaluating single- and double- elective embryo transfers in oocyte donation cycles reported similar results [22]. However, it remains controversial how many embryos should be transferred after initial failure of eSBT.

Only one related study, by Monteleone et al. [23], evaluated pregnancy outcomes in patients who failed to conceive in fresh eSET cycles and subsequently underwent either elective double vitrified-warmed blastocyst transfer or elective single vitrified-warmed blastocyst transfer. They reported similar pregnancy rates between the two groups but a higher multiple pregnancy rate in the double transfer group (22.5% vs. 5.9%), consistent with our results. Accordingly, ESHRE guidelines recommend that the decision to perform double embryo transfer instead of eSET should not be based on the number of previous unsuccessful ART treatments [8], given the significantly higher risk of multiple pregnancies and associated obstetric and perinatal complications. However, Monteleone et al. [23] only included blastocyst transfers, and the clinical pregnancy rate in their fresh eSBT cycles was relatively low (24.8%), suggesting that the study population may not have had good prognoses, thus limiting the reliability of their findings. In contrast, our results indicate that in patients with good prognosis, eSBT is preferable to double high-quality cleavage embryo transfer, as it reduces multiple pregnancy risk without compromising pregnancy outcomes.

In our study, the implantation rate was significantly lower in double cleavage embryo transfer, which is expected because some cleavage embryos fail to develop into blastocysts. Extended embryo culture serves as a selection process, which explains why clinical pregnancy and ongoing pregnancy rates were similar between groups. Our findings align with previous studies comparing double cleavage embryo transfer and SBT. Long et al. [24] reported no statistical difference in clinical pregnancy rates between double cleavage embryo transfer and SBT, although the live birth rate was slightly lower in the SBT group (23.0% vs. 29.0%; aOR: 0.78; 95% CI: 0.72–0.85). However, the double cleavage embryo transfer group had higher risks of twin births, preterm births, low birth weights, and small-for-gestational-age infants. Similarly, Wei et al. [25] found that transferring two high-quality 8-cell cleavage embryos achieved comparable clinical pregnancy and live birth rates to low-quality blastocysts but resulted in higher multiple pregnancy rates. Several other studies reached similar conclusions [26, 27], although they did not account for prior FET failure. Given the significantly higher risk of multiple pregnancies after double embryo transfer, we recommend SBT in good-prognosis patients even after previous ART failures. Furthermore, our findings suggest that freezing two high-quality cleavage embryos may be unnecessary in such patients, and extended blastocyst culture of all cleavage embryos should be considered. Gingold et al. [28] reported that nonadherence to ASRM guidelines, which recommend eSET in patients with favorable prognoses, occurred in 42%–45% of cases. This nonadherence resulted in multiple live birth rates of around 40%, thereby increasing maternal risk and healthcare costs [15, 28]. Patient education, as well as improved adherence by reproductive endocrinology and infertility specialists, may help increase eSET uptake.

When selecting an embryo transfer strategy after initial failure, cost-effectiveness should also be considered. The primary concerns are the costs of extended embryo culture versus those associated with complications from multiple pregnancies. A systematic review comparing double embryo transfer with sequential SET [29] reported comparable cumulative live birth rates but significantly lower multiple pregnancy risks with sequential SET, albeit with longer treatment times. While sequential SET theoretically increases treatment costs by requiring more cycles, the reduced complications from multiple pregnancies may offset these expenses. In our study, the cost of extended embryo culture was lower than the potential costs associated with multiple pregnancy complications. Thus, extended embryo culture is preferable to the risks of multiple pregnancy. However, patients must also be informed of the risk that no embryos may remain available after extended culture, which could increase psychological stress.

This study has certain limitations. All data were derived from a single fertility center, and the retrospective design may have introduced confounding factors. Therefore, caution is required when generalizing these findings to broader populations with varied prognoses.

Single blastocyst transfer achieves similar pregnancy outcomes as double, high-quality cleavage embryo transfer but carries a lower risk of multiple pregnancies following failed single blastocyst transfer. Larger prospective randomized trials are needed to confirm these findings.

All data generated or analyzed during this study are included in this article. Further inquiries can be directed to the corresponding author.

SL and CZL designed the present study. SL, ZXW and BLS collected raw data. SL and DGL checked and analyzed all data. SL were major contributors in writing the manuscript. CZL revised the manuscript. All authors contributed to editorial changes in the manuscript. All authors read and approved the final manuscript. All authors have participated sufficiently in the work and agreed to be accountable for all aspects of the work.

The study was carried out in accordance with the guidelines of the Declaration of Helsinki. A waiver of informed consent was obtained for this study because it did not disclose any personal or identifying patient information and posed no risk to the participants. Approval was granted by the Research Ethics Committee of the Peking University Shenzhen Hospital(approval number: 2024-133).

This work was supported by grants from Sanming Project of Medicine in Shenzhen (No. SZSM202211043), National Key Research and Development Program (No. 2024YFC2707503), Sanming Project of Cervical Disease Prevention and Control from Pingshan District Maternal and Child Health Hospital and Shenzhen Peking University Shenzhen Hospital (No. 2023-1).

The authors have no conflicts of interest to declare.