Double Embryo Transfer vs. Single Blastocyst Transfer in Frozen-Thawed Cycles: A Retrospective Comparative Study

Lim YH, Sim PK, Nadkarni P. A retrospective comparative study of double cleavage-stage embryo transfer versus single blastocyst in frozen-thawed cycles. Middle East Fertility Society Journal 2024;29:17. https://doi.org/10.1186/s43043-024-00177-7

---

Received: 22 November 2023  |  Accepted: 12 March 2024  |  Published online: 22 March 2024  |  Last reviewed: July 2026

This page summarises original clinical research conducted at KL Fertility & Gynaecology Centre and published in a peer-reviewed journal. It is presented here for the clinical and scientific community and is not intended as a substitute for individualised patient counselling.

Funding and disclosures: No specific grant was received from public, commercial, or not-for-profit funding bodies for the conduct of this study. Medical writing assistance for the original publication was supported by Firstline Pharmaceuticals Sdn Bhd. The authors report no competing interests.

Data availability: The datasets analysed during this study are available from the corresponding author on reasonable request.

In This Research

• Background and Rationale
• Study Design and Methods: Day 3 DET vs. Single Blastocyst Transfer in FET Cycles
• Results: Implantation, Pregnancy, and Multiple Pregnancy Rates
• Discussion: How These Findings Compare With Published Evidence
• Clinical Practice Implications
• Limitations of This Retrospective Cohort Study
• Key Findings
• About the Researchers
• Download the Original Research
• References

Background and Rationale

Multiple embryo transfer has long been used to increase the likelihood of live birth in IVF, but it carries a well-documented cost: higher rates of multiple pregnancy, with attendant maternal and neonatal complications, including miscarriage, fetal death, fetal malformation, and pregnancy complications compared with singleton pregnancies. Multiple pregnancies have historically resulted from the difficulty of identifying the single most competent embryo at the day 2/3 cleavage stage, leading clinicians to transfer more than one embryo to preserve pregnancy rates.

Extended culture to the blastocyst stage has gained traction as an alternative, since it allows more reliable selection of a single competent embryo and has been associated with reduced multiple pregnancy risk and improved clinical outcomes relative to cleavage-stage transfer. However, at the time of this study, no clinical guideline or consensus existed specifically comparing blastocyst-stage and cleavage-stage transfer outcomes. A 2022 consensus statement on embryo transfer addressed technical aspects of the procedure but not this comparison, and a 2022 Cochrane review concluded that more evidence was needed to determine the effect of transfer stage on cumulative live birth and pregnancy rates.

This study aimed to compare success rates between day 3 double cleavage-stage embryo transfer (DET) and single blastocyst transfer (SBT) in women undergoing frozen embryo transfer (FET) across three endometrial preparation protocols, namely natural cycle (NC), modified natural cycle (m-NC), and hormone replacement therapy (HRT), at a single fertility centre in Malaysia.

Study Design and Methods: Day 3 DET vs. Single Blastocyst Transfer in FET Cycles

Design: Retrospective cohort study.

• Design: Retrospective cohort study.
• Subjects: 999 female patients under 38 years of age who underwent day 3 DET or SBT during FET cycles at KL Fertility & Gynaecology Centre, Malaysia, from January 2019 to December 2021. The cohort included patients with primary and secondary subfertility, male factor infertility, tubal factor infertility, polycystic ovary syndrome, and endometriosis.
• Exclusion criteria: Gamete donation, embryo donation, cycles involving preimplantation genetic testing, assisted hatching, and cases with incomplete data (including overseas patients or those referred from other centres). All patient data were de-identified. No informed consent was required given the retrospective design of the study.
• Fertilisation and embryo culture: All oocytes were inseminated by intracytoplasmic sperm injection (ICSI) 38-40 hours post-hCG administration. Fertilisation was assessed on day 1. Embryos were graded and examined on day 3 and day 5/6/7. Embryo culture was performed at 37°C under 6% CO₂, 5% O₂, and 89% N₂ using sequential culture media (COOK, Sydney IVF) in a benchtop incubator (Origio/Planer BT37).
• Embryo grading: Day 3 cleavage-stage embryos were graded 1-4 based on cell number, blastomere symmetry, and fragmentation (Grade 1: 6-10 cells, even blastomeres, no fragmentation; Grade 4: <4 cells, uneven blastomeres, >25% fragmentation). Day 5/6/7 blastocysts were graded A-D for inner cell mass (ICM) and trophectoderm quality.

Study groups:

• Day 3 DET (n=274): Two grade 1-2 cleavage-stage embryos, vitrified and thawed for transfer.
• SBT (n=725): One blastocyst of grade BC, CB, or above (full, expanded, hatching, or fully hatched blastocyst with ICM/trophectoderm grades A-C), vitrified and thawed for transfer.
• Vitrification and thawing: Cryotop method using Kitazato vitrification and thawing media (Kitazato Corporation, Japan), per Kuwayama (2007).
• Endometrial preparation: FET cycles were performed following NC (n=400), m-NC (n=243), or HRT (n=356) protocols, with luteal phase support via oral dydrogesterone 10 mg twice daily (NC and m-NC groups) or vaginal micronized progesterone (Utrogestan 200 mg three times daily, HRT group). Full protocol details are available in the original publication.

Outcome measures:

• Positive pregnancy: hCG >25 IU/L on day 10 or 12 post-transfer.
• Clinical pregnancy: intrauterine sac confirmed on transvaginal ultrasound at 5 weeks post-transfer.
• Implantation rate: implanted embryos (sacs at 6 weeks) per embryo transferred.
• Multiple pregnancy rate: two or more gestational sacs at 12 weeks, expressed per clinical pregnancy.
• Ongoing pregnancy rate: pregnancy reaching 12 weeks’ gestation.
• Statistical analysis: SPSS v26.0. Chi-square testing for categorical variables; one-way ANOVA for continuous variables. Significance threshold: p < 0.05. Baseline age did not differ significantly between groups (DET 33.2 ± 2.9 years vs. SBT 32.9 ± 2.9 years; p = 0.183).

Results: Implantation, Pregnancy, and Multiple Pregnancy Rates

Table 1. Overall success rates, DET vs. SBT (per embryo transfer cycle)

No statistically significant differences were observed between groups in positive pregnancy, clinical pregnancy, or ongoing pregnancy rates. Implantation rate was significantly higher with SBT (50.1% vs. 37.6%, p < 0.001). Multiple pregnancy rate was significantly and substantially lower with SBT (1.1% vs. 28.7%, p < 0.001).

Table 2. Implantation and multiple pregnancy rates by endometrial preparation subgroup

The implantation-rate advantage for SBT reached statistical significance in the NC and HRT subgroups but not in the m-NC subgroup, though the direction of effect was consistent across all three. The reduction in multiple pregnancy rate with SBT was statistically significant across all three endometrial preparation protocols.

Discussion: How These Findings Compare With Published Evidence

The higher implantation rate observed with SBT is consistent with prior literature showing improved implantation rates for blastocyst-stage relative to cleavage-stage transfer, plausibly attributable to more effective embryo selection following extended culture to day 5.

The substantially lower multiple pregnancy rate with SBT (1.1% vs. 28.7%) is similarly consistent with previously reported findings favouring SBT for multiple-pregnancy risk reduction, though prior cohorts studying this question generally skewed younger (under 36 years) than the present cohort (up to 38 years).

No significant difference was found between groups in positive pregnancy, clinical pregnancy, or ongoing pregnancy rates, indicating that comparable pregnancy outcomes are achievable with either approach, a finding broadly in line with the wider literature. One point of divergence is worth noting: at least one other retrospective analysis found that day 6 SBT specifically outperformed both day 3 DET and day 5 SBT on pregnancy rate. The present study did not stratify by blastocyst day (5 vs. 6/7), and so cannot confirm or refute that finding.

Current professional guidance has moved toward single embryo transfer more broadly. The European Society of Human Reproduction and Embryology’s 2024 guideline recommends single embryo transfer for women of essentially all ages, given the added maternal and fetal risks associated with multiple pregnancy. The American Society for Reproductive Medicine has similarly narrowed recommended embryo numbers across successive guideline revisions. The present findings are consistent with, and add single-centre Malaysian cohort data in support of, that direction of practice.

Clinical Practice Implications

Taken together, these findings support single blastocyst transfer as the preferred default strategy for frozen embryo transfer in patients with an available good-quality blastocyst, on the basis that it achieves implantation and pregnancy outcomes at least comparable to day 3 double embryo transfer while substantially reducing multiple pregnancy risk, a reduction that held across all three endometrial preparation protocols studied.

This has particular relevance for HRT-cycle patients, who are independently associated with worse obstetric and perinatal outcomes in the broader literature; avoiding multiple pregnancy in this subgroup carries added weight when protocol and transfer strategy are being selected jointly.

Where day 3 DET may still warrant individualised consideration includes cases without a blastocyst-stage embryo available for transfer, or where embryo number or quality constraints limit extended culture, circumstances the present dataset does not directly address, since the comparison was between embryos that had already reached each respective stage.

Limitations of This Retrospective Cohort Study

• Retrospective, non-randomised design: allocation to DET or SBT followed physician judgement and patient preference rather than random assignment, introducing potential selection bias.
• Comparisons were made between different women rather than within the same woman receiving both transfer types.
• Day 5, 6, and 7 blastocysts were not analysed separately, despite evidence that outcomes may differ by blastocyst day.
• Maternal BMI, smoking status, alcohol use, and semen quality, all factors with documented potential influence on neonatal and blastocyst-formation outcomes, were not collected.
• The cohort was restricted to women under 38 years; findings may not generalise to older patients.
• As a single-centre study, results reflect one centre’s clinical and embryology protocols, which may limit generalisability, though this also removed inter-centre variation in culture media and technique as a confound.

Key Findings

• In 999 women undergoing FET (274 DET, 725 SBT), positive, clinical, and ongoing pregnancy rates did not differ significantly between day 3 DET and SBT.
• Implantation rate was significantly higher with SBT (50.1% vs. 37.6%, p < 0.001).
• Multiple pregnancy rate was significantly and substantially lower with SBT (1.1% vs. 28.7%, p < 0.001).
• The multiple-pregnancy-rate advantage for SBT held across all three endometrial preparation protocols (NC, m-NC, HRT).
• Findings support SBT as the preferred transfer strategy where a suitable blastocyst is available, consistent with current ESHRE and ASRM guidance favouring single embryo transfer.

About the Researchers

• Dr. Yun-Hsuen (Helena) Lim is a Fertility Specialist and Consultant in Obstetrics & Gynaecology at KL Fertility & Gynaecology Centre. She is a Fellow of the Royal College of Obstetricians and Gynaecologists (UK), holds a Master’s degree from Universiti Kebangsaan Malaysia, and was formerly a Senior Lecturer in the Department of Obstetrics & Gynaecology at Universiti Kebangsaan Malaysia. She is also a Clinical Associate Professor at Universiti Tunku Abdul Rahman, Malaysia. She was the primary author responsible for study design and manuscript preparation. (ORCID: 0000-0003-4153-3033)
• Poo Keen Sim is a member of the Scientific Team and the Chief Embryologist at KL Fertility & Gynaecology Centre, with over 20 years of hands-on experience in clinical embryology. She holds a Master’s degree in Clinical Embryology and was responsible for data collection for this study.
• Dato’ Dr. Prashant Nadkarni is a Fertility Specialist in Reproductive Medicine at KL Fertility & Gynaecology Centre. He was responsible for reviewing and revising the manuscript.

Acknowledgement (per original publication): The authors thank the clinical, nursing, and embryology teams of KL Fertility Centre for their contributions to this study, and Anne John Michael for medical writing assistance.

Download the Original Research

Download PDF Now

References

1. Rao J, Qiu F, Tian S, Yu Y, Zhang Y, Gu Z, Cai Y, Jin F, Jin M. Clinical outcomes for Day 3 double cleavage-stage embryo transfers versus Day 5 or 6 single blastocyst transfer in frozen-thawed cycles: a retrospective comparative analysis. J Int Med Res 2021;49(12). https://doi.org/10.1177/03000605211062461
2. Glujovsky D, Quinteiro Retamar AM, Alvarez Sedo CR, Ciapponi A, Cornelisse S, Blake D. Cleavage-stage versus blastocyst-stage embryo transfer in assisted reproductive technology. Cochrane Database Syst Rev 2022;Issue 5:CD002118. https://doi.org/10.1002/14651858.CD002118.pub6
3. ESHRE Guideline Group on the Number of Embryos to Transfer. ESHRE guideline: number of embryos to transfer during IVF/ICSI. Hum Reprod 2024;39(4):647-663. https://academic.oup.com/humrep/article/39/4/647/7607249
4. Practice Committee of the American Society for Reproductive Medicine. Guidance on the limits to the number of embryos to transfer: a committee opinion. Fertil Steril 2021;116:651-654.
5. Lim YH, Sim PK, Nadkarni P. A retrospective comparative study of double cleavage-stage embryo transfer versus single blastocyst in frozen-thawed cycles. Middle East Fertility Society Journal 2024;29:17. https://doi.org/10.1186/s43043-024-00177-7