Embryo cryopreservation has become a cornerstone of assisted reproductive technologies (ART). Initially developed to preserve surplus embryos after fresh in vitro fertilization and embryo transfer (IVF-ET) cycles, frozen embryo transfer (FET) has been shown to be associated with superior clinical outcomes in certain patient populations. Controlled ovarian stimulation disrupts normal endometrial physiology and receptivity, leading to impaired receptivity and embryo-endometrial asynchrony [1, 2]. In contrast, FET cycles better mimic natural endocrine conditions, thereby restoring endometrial receptivity and improving synchrony [3, 4]. The ‘freeze-all’ strategy further optimizes outcomes in high responders and reduces the treatment burden by avoiding repeated ovarian stimulation. Particularly for older patients with diminished ovarian reserve, FET can effectively mitigate the detrimental impact of supraphysiologic stimulation on endometrial receptivity, thereby increasing the likelihood of a live birth [5]. Nevertheless, optimizing pregnancy rates after FET remains a significant challenge in reproductive medicine.

Vitrification can be used to preserve embryos at different developmental stages, and the survival rate of warmed embryos is closely associated with the developmental stage. Although current methods for assessing embryonic developmental potential remain limited, advances in culture systems have improved the evaluation of viability through extended in vitro culture, which helps to avoid unnecessary transfers and reduces intrauterine interventions. Consequently, selecting the optimal developmental stage for cryopreservation is critical for maximizing ART success. Currently, the most commonly cryopreserved stages are cleavage-stage (Day 3) and blastocyst-stage (Day 5 or Day 6) embryos, with most studies focusing on the extended culture from cleavage-stage embryos to blastocysts or overnight culture before transfer [6, 7, 8, 9]. Blastocyst-stage transfers have been shown to yield better clinical outcomes than do cleavage-stage embryo transfers [6, 10, 11]. However, practical considerations such as patient-scheduling constraints or embryology-laboratory schedules often necessitate cryopreservation at alternative times (Day 2 or Day 4) [12, 13], a practice reflected in Department of Human Reproductive Medicine, Beijing Obstetrics and Gynecology Hospital, Capital Medical University where embryos are cryopreserved from Day 2 to Day 6.

Although some studies have been done on this topic [14, 15], no previous study has systematically analyzed a substantial number of cases across these developmental periods. In the present large-scale retrospective study, we analyzed 9981 frozen-warmed embryos cultured for varying durations (Day 2 to Day 5). The choice of freezing timing is influenced by multiple factors (e.g., laboratory and clinical scheduling, weekend adjustments), particularly on Day 2 and Day 4. We performed a detailed stratification based on embryo-warming day and transfer timing to systematically evaluate their impact on clinical outcomes and to identify optimal transfer strategies.

To our knowledge, this study was the first to compare pregnancy outcomes after cryopreservation at multiple developmental stages [cleavage-stage (including Days 2 and 3), morula, and blastocyst] in a large patient cohort. Our research demonstrated that embryos warmed on Day 2 and subsequently cultured to Day 3 exhibited significantly higher clinical-pregnancy and live-birth rates. Furthermore, embryos warmed on Day 3 and cultured to Days 4 or 5 also showed better clinical outcomes. No significant differences were observed in clinical outcomes among groups when all embryos were cultured to Day 5, irrespective of the original warming day.

In clinical practice, cleavage-stage embryo transfer is typically performed on Day 3, yet no consensus exists on whether transfer on Day 2 or Day 3 yields the better outcome. A previous study suggested that Day-3 transfers are associated with lower survival rates and higher miscarriage rates than were Day-2 transfers; they attributed this to damage during freeze-thaw procedures [18]. That led to the hypothesis that Day-2 embryos may tolerate freeze-thaw procedures better. However, some studies reported no statistical difference in clinical outcomes when Day-2 warmed embryos underwent overnight culture [14, 19]. With advancements in vitrification techniques, freeze-warm-related damage has been substantially minimized. A study has indicated that transferring embryos with $\ge$50% blastomere survival post-warming did not influence neonatal outcomes [12]. Nevertheless, our data indicated that overnight culture of Day-2-warmed embryos (to Day 3) produced better clinical outcomes than did same-day transfer, contradicting some prior conclusions. One study showed a negative correlation between the degree of freeze-warm damage and the number of blastomeres [18]. Furthermore, there is a significant association between the rate of overnight cleavage and positive hCG rates, as well as implantation rates [12]. Extended culture allows for functional assessment of post-warming embryo viability, enabling the exclusion of developmentally arrested embryos—often linked to molecular defects and genetic abnormalities [20]. Ottolini et al. [21] linked early cleavage issues with developmental arrest, noting that failure to coordinate cell cycle and centrosome duplication caused pre-implantation embryonic arrest in vitro. High chromosomal aneuploidy rates in embryos with poor developmental potential increased implantation failure and miscarriage rates [22]. Notably, embryonic arrest most frequently occurs at the 4-8-cell stage [23]. Further blastomere growth may also improve embryo-endometrium synchrony [24]. Another study showed that Day 3 FET showed a better clinical outcome than did Day 2 FET, and did not affect embryo survival [25], which is consistent with our findings. Therefore, we recommend culturing Day-2 warmed embryos to Day 3 before transfer, as this allows for selection of embryos with better developmental potential and is associated with higher clinical pregnancy rates.

Morula-stage embryo transfer represents a clinically feasible strategy, though it remains underutilized in ART. After fertilization, embryos undergo compaction typically at the 8-cell stage, forming a morphologically distinct morula—a transitional phase between cleavage and blastocyst stages. Characterized by a tightly packed cellular mass, the morula is often perceived as fragile due to its lack of clear morphological markers [26]. This morphological ambiguity, coupled with stage-specific biological complexity, has limited clinical research on morula transfer, resulting in its frequent oversight as a valuable transfer option [13]. At this developmental juncture, embryos exhibit heightened DNA-synthesis activity, priming for subsequent cell-lineage differentiation critical for implantation [27]. The compaction process involves multiple self-correcting mechanisms that may enhance developmental competence [28]. One study showed that there was no statistical difference in clinical pregnancy outcomes for embryos frozen on Day 3, whether they were transferred after warming or after overnight culture [8]. Our study indicated that extended culture of thawed cleavage-stage (Day 2/3) embryos to morula stage (Day 4) was associated with better clinical outcomes. This association was significantly stronger for embryos thawed on Day 3 compared to those thawed on Day 2. No significant differences in clinical pregnancy rates have been shown to result from embryos transferred on Day 4 and Day 5 [13, 29]. Our findings align with those observations. Our study showed that there were no significant differences in clinical outcomes between embryos transferred on Day 4 immediately after warming and those transferred after overnight culture. However, A study suggested that Day-5 transfers have higher success rates than Day-4 transfers [30]. It is possible that Day 4 transfer is often not based on clinical indications, but on logistical limitations such as public holidays, or scheduling issues. This nonrandom selection introduces potential bias. Therefore, the research on Day 4 transfer is relatively limited, and further research is needed.

The transfer of blastocysts has been reported to have better clinical outcomes than does the transfer of cleavage-stage embryos [31, 32]. Our experiments showed that although extended culture to Day 5 was associated with better outcomes, this association was significant only for embryos thawed on Day 3, not for those thawed on Day 2. Early embryo development primarily reflects oocyte quality, which does not fully predict subsequent embryo development. During development into a blastocyst, the genome regulates the transitions from maternal to zygote control [33]. Blastocysts, with their higher cell numbers and structured organization (ICM and TE), offer enhanced viability and more reliable implantation potential than do cleavage-stage embryos [15]. With the success of blastocyst culture, chromosomal abnormalities become less frequent, facilitating improved screening of suitable embryos for transfer. Physiologically, embryos with 4–8 cells initially reside in the fallopian tubes, entering the uterus upon reaching the blastocyst stage. Therefore, transferring blastocysts rather than cleavage-stage embryos may align more closely with the natural timing of the embryo to enter the uterus. Better synchronization with the endometrial-receptivity window may potentially improve pregnancy rates [31, 34].

The present study showed that no significant differences exist between transferring frozen-warmed blastocysts and transferring blastocysts cultured from warmed cleavage-stage embryos. This was consistent with another study showing that extending culture of cleavage-stage embryos to blastocysts is comparable to using frozen-warmed blastocysts, with similar live-birth rates [35]. For patients with recurrent implantation failure who have blastocysts available, prioritizing frozen-warmed blastocyst FET is recommended. Otherwise, in cases without blastocyst preservation, extended culture of frozen-warmed Day 3 embryos to Day 5 for blastocyst transfer is advisable, as both strategies yield similar pregnancy outcomes [10]. However, extending embryo culture without selection is not beneficial, as it increases the transfer-cycle cancellation rate and reduces the cumulative pregnancy rate [36]. Some embryos may fail to develop into blastocysts in vitro, but they may develop and implant in vivo. For patients with diminished ovarian reserve or a strong desire for expedited treatment, early transfer/freezing (Day 2/3) may optimize embryo availability.

Although an extended post-warming culture entails additional costs and the risk of embryo attrition, it offers significant advantages by eliminating developmentally compromised or genetically abnormal embryos, thereby selecting those with higher implantation potential. This selective approach reduces the financial and emotional burdens associated with transferring non-viable embryos. Furthermore, extended culture may decrease ectopic-pregnancy risks by minimizing the embryo transit time within the uterine cavity. Clinicians should therefore adopt a patient-centered approach when recommending extended culture, carefully weighing individual circumstances and preferences.

Selective cryopreservation and FET are safe and effective strategies for optimizing cumulative live birth rates in ART. After adjustment for confounding factors, a significant correlation was found between extended embryo culture and superior clinical outcomes. The statistical significance was particularly strong in two groups: embryos thawed on Day 2 and cultured to Day 3, and those thawed on Day 3 and cultured to Day 4 or 5. Direct transfer of frozen-warmed blastocysts and transfer of blastocysts cultured from warmed cleavage-stage embryos both achieve comparable clinical success. Furthermore, when logistically necessary (e.g., due to clinic or patient scheduling constraints), Day 4 transfer can also achieve comparable clinical outcomes. Therefore, individualized extended culture strategies were associated with FET outcomes. However, if no blastocysts are available, cleavage-stage embryo transfer should be considered in subsequent cycles to maximize reproductive potential.

The data used and analyzed during the current study are available from the corresponding authors on reasonable request.

Study design was proposed by ZX, XY and QW. Material preparation and data collection were performed by LY, RS and LW. Data check was done by YF and QW. Data analysis and the first draft of the manuscript were written by QW. 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. The study protocol was approved by the Ethics Committee of Beijing Obstetrics and Gynecology Hospital, Capital Medical University (date: January 30, 2024. No. 2024-KY-024-01). The ethics committee granted a waiver of informed consent for this retrospective study, given the use of anonymized data and the absence of risk to individuals.

We would like to express our gratitude to all those who helped us during the writing of this manuscript.

This study was supported by the Beijing Hospitals Authority’ Ascent Plan (DFL20191401) and Specialized Youth Foundation Project of Beijing Obstetrics and Gynecology Hospital, Capital Medical University (FCYYQN-202104).

The authors declare no conflict of interest.