Introduction
Recent advancements in assisted reproductive technology (ART) have focused on improving efficiency, reducing the duration of reproductive management, and promoting a more patient-centered approach [1,2]. Patients undergoing in vitro fertilization (IVF) are increasingly involved in clinical decision-making compared with patients in many other medical fields [3]. This shift has led to greater emphasis on evidence-based and data-driven decision-making in reproductive medicine. Contemporary infertility treatments incorporate strategies such as freeze-all approaches, single embryo transfer (SET), extended embryo culture, and pre-implantation genetic testing for aneuploidy (PGT-A) to optimize pregnancy success rates while respecting patient autonomy [4-8].
Not all embryos progress to fully expanded blastocysts, making the identification of embryos with the highest developmental potential critical for successful IVF outcomes. However, the selection process remains challenging due to ongoing debate regarding which embryonic characteristics best reflect this potential. Some studies report no definitive association between embryo morphology and developmental competence, suggesting limited predictive value for morphological assessment [9,10], whereas other studies demonstrate a stronger correlation between morphological characteristics and clinical outcomes [11,12]. Determining whether morphological attributes observed during early blastocyst development can reliably predict clinical outcomes therefore remains an important objective [13]. Embryonic development to the blastocyst stage is closely associated with pregnancy success. Morphological assessment remains the most fundamental and non-invasive approach for identifying high-quality embryos and is widely used as an indicator of implantation potential and successful pregnancy [14]. Accordingly, identifying developmental parameters with strong predictive value for morphological selection is essential, and selecting embryos on the basis of these parameters is critical for optimizing clinical outcomes [3,15]. In this study, we aimed to establish more definitive criteria for embryo selection.
Evaluating the combined influence of blastocyst quality and blastulation speed on pregnancy outcomes is of direct relevance to clinical practice. Despite extensive investigation, existing studies report conflicting results and ongoing disagreement regarding the optimal parameters for predicting embryo competence [16-18]. Most prior research, including our own, has focused predominantly on the developmental speed of cryopreserved blastocysts, with comparatively limited attention to detailed morphological grading [13,19-21].
In response to unresolved questions from previous investigations, the present study specifically examined the relative contributions of blastocyst quality and blastulation speed. We performed a comprehensive analysis of clinical outcomes to evaluate how these factors independently and jointly influence pregnancy outcomes. Through this detailed assessment, we sought to clarify the relative importance of each parameter, provide a more nuanced understanding of their respective roles, and ultimately improve the accuracy of embryo competence assessment. This approach is intended to support more informed clinical decision-making and to advance personalized practices in ART.
Methods
1. Ethics statement
The study protocol was approved by the Institutional Review Board (IRB) of CHA Gangnam Medical Center (IRB approval number: GCI IRB 2024-07-017-001) and was conducted in accordance with the principles of the Declaration of Helsinki. Given the retrospective nature of the study, the requirement for informed participant consent was waived, and only medical records were used for analysis. The study was conducted in strict compliance with all relevant guidelines and regulations.
2. Study design and patient cohort
This retrospective cohort study evaluated outcomes from 736 single vitrified-warmed blastocyst transfer (SVBT) cycles performed at the Gangnam CHA Fertility Center in South Korea between January 2019 and December 2021. From an initial total of 2,134 vitrified-warmed embryo transfer (ET) cycles, ineligible cases were excluded according to the following criteria: (1) embryos originating from other hospitals (n=66); (2) cycles without ET or those canceled prior to transfer (n=57); (3) use of double or multiple embryos for warming and/or transfer (n=1,190); (4) use of pronuclear, cleavage-stage, or morula-stage embryos (n=56); (5) uterine anomalies that impeded transfer, cases involving embryo shrinkage, or transfers involving day 7 frozen blastocysts (n=20); and (6) missing data or follow-up limitations (n=9). In addition, a retrospective analysis was performed to evaluate the euploidy status of 395 embryos derived from 171 fresh IVF cycles that underwent PGT-A between January 2022 and May 2023. The study flowchart is presented in Figure 1.
3. Blastocyst quality evaluation and grading
Blastocyst quality assessment was performed as described in our previous studies [22,23]. Briefly, blastocyst morphological scores were determined using the Blastocyst Quality score (BQS), which integrates multiple morphological parameters, including blastocyst expansion and the cell numbers of the inner cell mass (ICM) and trophectoderm (TE). The BQS was calculated as the product of the blastocoel expansion score, ICM score, and TE score (BQS=blastocoel expansion score×ICM score×TE score). Based on BQS values, embryos were classified into four groups with the following criteria and grade distributions: (1) top group (score, 24–45): expanded AA, hatching AA, hatching AB/BA, hatched AA, and hatched AB/BA; (2) good group (score, 16–23): mid AA, expanded AB/BA, hatching BB, and hatched BB; (3) average group (score, 7–15): early AA, mid AB/BA, mid BB, expanded BB, expanded AC/CA, hatching AC/CA, hatching BC/CB, hatched AC/CA, and hatched BC/CB; and (4) poor group (score, 1–6): early AB/BA, early BB, early AC/CA, early BC/CB, early CC, mid AC/CA, mid BC/CB, mid CC, expanded BC/CB, expanded CC, hatching CC, and hatched CC.
4. Blastocyst vitrification and warming procedures
Blastocyst vitrification and warming were performed using the slush liquid nitrogen (LN₂) and electron microscopy gold grid method, in accordance with procedures described in our previous studies [13,15]. Briefly, vitrification and warming were conducted in a standardized manner. The blastocyst was immobilized using a holding pipette, after which artificial shrinkage and assisted hatching were performed using the ZILOS-TK laser system (Hamilton Thorne Inc.). Following equilibration, vitrification was carried out using a solution containing ethylene glycol, dimethyl sulfoxide, 0.5% sucrose, and 20% human serum albumin in hydroxyethyl piperazine ethane sulfonicacid (HEPES) medium. Blastocysts were loaded onto an electron microscopy gold grid in a minimal volume (<0.1 μL) and immediately submerged in slush LN₂ using a Vit-Master (IMT International) device.
One day before ET, vitrified blastocysts were warmed to 37 °C and sequentially transferred into warming solutions containing 0.5, 0.25, 0.125, and 0.0 M sucrose at 2.5-minute intervals. Warmed blastocysts were subsequently cultured in an incubator maintained at 37 °C with 6% CO₂, 5% O₂, and 89% N₂ until transfer. The survival rate of vitrified blastocysts was 97.7%, with 873 of 894 blastocysts surviving from 736 SVBT cycles. Embryo survival and viability were systematically evaluated at multiple time points in accordance with previously established protocols [13]. Viability was initially assessed within 2 hours after warming. Re-expansion and degeneration were evaluated the following morning during ET preparation, with a final assessment performed immediately prior to ET. Blastocyst morphology was evaluated independently by three experienced senior embryologists, who double-checked and reviewed the results while remaining blinded to clinical outcomes (Supplementary Methods, Supplementary Table 1, and Supplementary Figure 1).
5. Blastocyst TE biopsy and PGT-A procedures
Blastocyst TE biopsy and PGT-A procedures were conducted according to standardized protocols described previously [23,24]. Briefly, TE biopsy was performed on day 5 or day 6 using micromanipulators and an inverted microscope TE-2000 (Nikon Instruments Inc.). The blastocyst was stabilized with a holding pipette, and the zona pellucida was punctured using the ZILOS-TK laser system. A biopsy pipette positioned on the opposite side was then used to gently aspirate TE cells through the opening, thereby separating them from the zona pellucida. The biopsied TE cells were washed in D-phosphate-buffered saline (PBS) (PBS without Ca²⁺ and Mg²⁺) and transferred to RNase–DNase-free polymerase chain reaction tubes containing 2 μL of PBS for genetic analysis at the CHA Biotech genetic analysis laboratory. Based on the genetic analysis results, embryos were classified as euploid, aneuploid, or mosaic.
6. Endometrial preparation in the SVBT cycle
Endometrial preparation for SVBT cycles followed established protocols described in our previous studies [25,26]. In brief, the endometrium was prepared using either natural cycles or hormone replacement cycles. Vitrified-warmed blastocysts were transferred using a COOK® ET catheter (COOK Medical) under transabdominal ultrasound guidance. Luteal phase support was initiated 5 days prior to ET and continued until the 10th week of gestation, using either vaginal progesterone suppositories or intramuscular progesterone injections.
7. Definitions of clinical outcome parameters and PGT-A classification
Primary clinical outcomes included serum beta-human chorionic gonadotropin (β-hCG) levels, implantation rate (IR), ongoing pregnancy rate (OPR), and miscarriage rate. Serum β-hCG levels were measured 11 days after ET, with values exceeding 20 mIU/mL indicating a positive pregnancy. The IR was defined as the number of gestational sacs per total number of transferred embryos and was used to calculate the clinical pregnancy rate (CPR) following single ET. An ongoing pregnancy was defined as a pregnancy with a detectable fetal heartbeat on ultrasound beyond 12 weeks of gestation. The miscarriage rate was defined as pregnancy loss occurring before 22 weeks of gestation. PGT-A was performed using a validated next-generation sequencing platform. Embryos were classified as euploid when <20% of analyzed cells exhibited chromosomal abnormalities, aneuploid when >80% of cells were abnormal, and mosaic when 20%–80% of cells were abnormal, in accordance with cutoff values recommended by the Preimplantation Genetic Diagnosis International Society.
8. Statistical analysis
Statistical analyses were performed using jamovi and GraphPad Prism ver. 10.0 software (GraphPad Software Inc.). Continuous variables are presented as mean±standard error of the mean and were analyzed using the Student’s t-test. Categorical variables were analyzed using the Pearson chi-square test. In addition, univariate and multivariate logistic regression analyses were conducted to evaluate associations between pre-vitrification and post-warming morphological factors. Statistical significance was defined as p<0.05.
Results
1. Characteristics of patients in SVBT
Table 1 summarizes the demographic characteristics of patients stratified by blastulation speed (day 5 or day 6). The two groups demonstrated comparable baseline characteristics, including maternal age, infertility etiology, endometrial thickness, and the protocol used for endometrial preparation (e.g., natural cycle or hormone replacement therapy).
2. Blastocyst quality post-warming procedures based on pre-vitrification quality
Blastocyst quality was evaluated both before vitrification and after warming using the BQS, and embryos were categorized into four groups to assess changes in morphological quality. As shown in Figure 2, top- and good-grade blastocysts largely maintained their morphological quality after warming, irrespective of blastulation speed. In contrast, day 6 average-grade blastocysts were significantly less likely than day 5 blastocysts to retain top or good grades following warming (39.0% vs. 69.2%, p<0.001). This finding indicates a higher likelihood of post-warming deterioration to average or poor grades among day 6 blastocysts compared with day 5 blastocysts (average: 35.6% vs. 21.0%, p=0.014; poor: 25.4% vs. 9.9%, p<0.001). Similarly, within the poor-grade group, day 6 blastocysts showed a greater tendency toward higher poor-grade outcomes after warming than day 5 blastocysts (37.3% vs. 23.7%, p=0.059), although this difference did not reach statistical significance.
3. Clinical outcomes of SVBT cycles
As illustrated in Figure 3, when blastulation speed was considered independently, day 5 blastocysts demonstrated a significantly higher OPR than day 6 blastocysts (48.7% vs. 39.1%, p=0.047). However, when blastocyst quality was taken into account, clinical outcomes for top- and good-quality blastocysts were comparable between day 6 and day 5, with no statistically significant differences observed in β-hCG positivity (62.7% vs. 66.7%, p=0.746), IR (54.2% vs. 61.1%, p=0.585), or OPR (49.2% vs. 55.6%, p=0.613). By contrast, among average- and poor-quality blastocysts, clinical outcomes—including β-hCG positivity, IR, and OPR—were significantly higher for day 5 blastocysts than for day 6 blastocysts (62.2% vs. 50.9%, p=0.028; 54.7% vs. 42.7%, p=0.023; and 48.6% vs. 36.5%, p=0.020, respectively).
Top- and good-quality embryos also demonstrated a higher proportion of hatching or hatched blastocysts after warming on day 6 compared with day 5 (94.4% vs. 75.5%, p=0.071), although this difference did not reach statistical significance. In contrast, average- and poor-quality embryos showed a lower proportion of hatching or hatched blastocysts after warming on day 6 than on day 5, with the difference reaching statistical significance in the average-quality group (71.2% vs. 82.9%, p=0.033) (Figure 4).
4. Analysis of chromosome euploidy rates
Figure 5 presents the results of aneuploidy analyses conducted between January 2022 and May 2023, encompassing 395 consecutive PGT-A tests in which a total of 395 blastocysts were biopsied. These analyses were derived from 171 fresh IVF cycles and were performed to assess genetic outcomes. Across all blastocyst quality groups, day 5 blastocysts exhibited a significantly higher euploidy rate than day 6 blastocysts (49.5% vs. 29.5%, p<0.001) (Figure 5A). However, no significant difference in euploidy rates was observed between day 5 and day 6 blastocysts classified as top- or good-quality (37.5% vs. 52.5%, p=0.439). In contrast, significant differences in euploidy rates were identified between day 5 and day 6 blastocysts graded as average quality (54.8% vs. 34.0%, p<0.005) and poor quality (40.7% vs. 16.2%, p<0.005), respectively (Figure 5B).
Discussion
1. Principal findings
This retrospective study evaluated the roles of blastulation speed and morphological quality in SVBT cycles with the aim of formulating a more effective strategy than current SET-based approaches. The results emphasize the importance of blastocyst quality assessment and optimal embryo selection, suggesting that a higher cutoff threshold for blastocyst grade may be appropriate when vitrification occurs on day 6 compared with day 5. Blastocysts exhibiting excellent morphological quality prior to vitrification, particularly those classified as top- and good-quality, were more likely to maintain or even improve their quality after warming, including day 6 embryos characterized by slower development. Overall, differences between day 5 and day 6 embryos were observed primarily among blastocysts of average or poor quality. As the use of SVBT cycles continues to increase and clinical efforts increasingly focus on maximizing the potential of all viable embryos while minimizing unnecessary wastage, these findings support the adoption of stricter grading standards for day 6 embryos to improve overall clinical outcomes. The principal findings of this study are summarized in Figure 6.
These findings underscore the need for a comprehensive evaluation of clinical outcomes associated with blastocyst selection criteria in vitrification. By jointly considering morphological quality, blastulation speed, and the integration of PGT-A, embryo viability may be predicted with greater accuracy.
2. Interpretation of study findings and comparison with current evidence in the literature
Blastulation speed and morphological quality are widely regarded as critical determinants of CPR in SVBT cycles. The present study sought to establish a tailored ET strategy that better aligns with contemporary clinical practice. Despite growing interest in optimizing SVBT protocols, the ideal timing and approach remain subjects of ongoing debate, and a clear consensus has yet to emerge. Furthermore, the existing literature addressing optimal strategies for achieving successful clinical pregnancy, particularly with respect to embryo developmental parameters, remains limited [15,27].
A comprehensive evaluation of SET outcomes requires the integration of multiple variables, including maternal age, blastulation dynamics, and blastocyst morphological grade. Although numerous studies have examined the influence of developmental stage at transfer, blastocyst quality has often not been analyzed as an independent determinant of clinical outcomes [17,28].
Several previous studies have reported that blastulation speed is significantly associated with clinical outcomes. Bourdon et al. [28] demonstrated that, in SVBT cycles, day 5 blastocysts result in significantly higher pregnancy rates than day 6 blastocysts, a finding supported by meta-analyses indicating improved cumulative pregnancy and live birth rates (LBRs). However, although developmental stage was evaluated, the influence of morphological quality was not incorporated. Zhang GL et al. [29] reported that poor-quality day 5 and high-quality day 6 blastocysts achieved comparable clinical outcomes, suggesting that blastulation speed may exert a stronger influence than morphology on implantation potential. Similarly, He et al. [17] recommended prioritizing the transfer of poor-quality day 5 blastocysts over high-quality day 6 blastocysts, reinforcing the hypothesis that earlier developmental progression confers an advantage .
In contrast, other investigations have demonstrated that when embryos of equivalent morphological grade are compared, day 5 and day 6 blastocysts exhibit similar euploidy rates, implantation potential, and clinical outcomes, with no significant differences in miscarriage or OPR [28,30,31]. Li et al. [27] further conducted stratified analyses based on blastocyst quality and reported no significant differences in CPR, LBR, or miscarriage rate between day 5 and day 6 blastocysts of comparable morphological grades.
Our findings are consistent with those reported by Li et al. [27] Specifically, when analyses were stratified by blastocyst quality, no significant differences in clinical outcomes were observed between high-quality day 5 and day 6 blastocysts. These results indicate that once morphological quality is adequately controlled for, the day of blastulation at vitrification or transfer may not independently determine clinical success.
The present study provides an objective evaluation of embryonic factors and highlights the importance of pre-vitrification embryo quality. Notably, top-quality embryos before vitrification were more likely to retain excellent morphology after warming, regardless of blastulation speed. In addition, our findings suggest that blastocyst quality, rather than the day of blastulation, plays a dominant role in determining post-warming developmental competence. Top-quality blastocysts demonstrated faster re-expansion following warming, which may contribute positively to clinical outcomes. In contrast, low-quality blastocysts exhibited reduced cell proliferation and metabolic activity and were more prone to morphological deterioration during the vitrification-warming process. These observations are consistent with findings reported in previous studies [3,13,15,32-34].
From a PGT-A perspective, slower-developing embryos have been reported to be associated with a higher prevalence of chromosomal abnormalities [35,36]. In addition, inferior blastocyst morphology may negatively affect euploidy status. Accordingly, simultaneous evaluation of embryo developmental speed and blastocyst morphology is essential for optimal embryo selection. Nevertheless, other investigators have reported minimal associations between blastocyst quality and LBR in frozen ET cycles, underscoring persistent controversy in this area [37,38]. In the present study, day 5 embryos demonstrated a higher euploidy rate than day 6 embryos (49.5% vs. 29.5%). However, no significant difference was observed between top-quality embryos on day 5 and day 6. In contrast, average- and poor-quality embryos showed divergent euploidy outcomes between the two blastulation days.
Our findings, consistent with previous research, reinforce the importance of selecting embryos with excellent morphological grades to achieve higher pregnancy rates. In both PGT-A and non–PGT-A cycles, blastocyst morphology significantly influences clinical outcomes after warming, whereas blastulation speed appears to have more limited relevance to pregnancy success and progression [28]. With the increasing adoption of PGT-A as a tool for optimizing embryo selection, morphological grading continues to play a central role in predicting pregnancy success [39,40]. Accordingly, ongoing efforts to enhance embryo quality are essential, not only to improve clinical outcomes but also to align patient expectations with realistic prognoses. Notably, this study provides a distinct contribution by incorporating both pre-vitrification and post-warming morphological assessments, thereby offering a more comprehensive evaluation of embryo quality than that reported in most previous studies.
3. Strengths and limitations
The principal strength of this study lies in its simultaneous evaluation of blastulation speed and embryo quality, combined with a detailed categorization across multiple quality grades for comparative analysis. This approach enabled the identification of meaningful differences in clinical outcomes between day 5 and day 6 embryos across quality strata, thereby clarifying which grades exhibited the most pronounced clinical variation. Moreover, even within the same morphological category, the wide range of scores suggests potential heterogeneity in true blastocyst quality among embryos, implying that conventional grading systems may not fully capture the intrinsic developmental competence of individual blastocysts.
Several limitations should also be acknowledged. First, the retrospective cohort design inherently limits control over potential confounding variables and introduces the possibility of selection bias. Second, the use of a single-center cohort may restrict the generalizability of the findings. In addition, the analysis was not confined exclusively to freeze-all cycles, which could introduce variability related to endometrial preparation protocols or timing of ET. Given these limitations, future research should include prospective, multi-center studies to validate the present findings under more controlled and diverse conditions. Future investigations should also explore the integration of automated embryo assessment technologies, such as artificial intelligence–based or time-lapse imaging systems, to improve grading consistency and reproducibility.
In conclusion, our findings demonstrate that blastocyst morphology plays a pivotal role in predicting clinical outcomes, underscoring the importance of careful morphological assessment when selecting embryos for vitrified-warmed SET cycles. For patients experiencing delayed embryo development, vitrification of high-quality, fully expanded day 6 blastocysts appears preferable to the transfer of low-grade or insufficiently expanded day 5 blastocysts. This strategy not only enhances clinical outcomes but also provides an objective means of evaluating blastocyst biological viability, ultimately supporting more personalized and effective embryo selection. Collectively, these insights offer practical guidance for clinicians and embryologists, enabling optimization of treatment strategies and improved management of patient expectations. Building on these findings, future research should further elucidate the underlying biological mechanisms—such as spindle integrity, mitochondrial dysfunction, mitochondrial DNA quantity, or metabolic delay—that may explain the association between delayed blastulation and reduced euploidy rates. Such efforts would strengthen the translational relevance of these results and contribute to the refinement of embryo selection strategies in clinical practice.
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