Relationships between morphological score changes during embryonic development and pregnancy outcomes
Article information
Abstract
Objective
This study investigated whether changes in embryonic morphological scores can predict pregnancy outcomes to support optimal blastocyst selection.
Methods
This retrospective dual-center study analyzed 1,492 single blastocyst transfers. Blastocysts were categorized according to day 3 and day 5 scores (excellent, good, or poor) and their day 3–5 morphological changes (improvement, stability, or deterioration). The primary outcomes were clinical pregnancy rate (CPR) and live birth rate (LBR).
Results
No statistically significant differences were observed in CPR or LBR among the Excellent-D3, Good-D3, and Poor-D3 embryo-quality groups. However, both CPR and LBR were significantly higher in the Excellent-D5 group compared with the Poor-D5 group (CPR: 58.64% vs. 46.34%, p=0.014; LBR: 45.97% vs. 33.54%, p=0.012). Temporal trends in developmental outcomes from day 3 to day 5 showed no significant differences across the improvement, stability, and deterioration groups for either CPR (57.95%, 55.28%, and 55.87%; p=0.615) or LBR (43.74%, 44.54%, and 40.84%; p=0.648). In stratified analysis, Poor-D3 embryos that reached Excellent-D5 status had a significantly lower LBR than Excellent-D5 embryos (40.52% vs. 48.81%: odds ratio, 0.714; 95% confidence interval, 0.513 to 0.994; p=0.046). No other subgroup comparisons demonstrated significant differences in clinical outcomes.
Conclusion
Day 5 morphology is the primary predictor of CPR and LBR, with Excellent-D5 embryos demonstrating superior outcomes. Although day 3–5 morphological changes were not associated with CPR or LBR, day 3 quality contributed supplemental predictive value for distinguishing among top-tier day 5 embryos, thereby aiding embryo selection.
Introduction
Currently, global infertility prevalence ranges from 12.6% to 17.5% [1], contributing to increasing reliance on in vitro fertilization and embryo transfer (IVF-ET). With ongoing advances in IVF-ET, the European Society of Human Reproduction and Embryology (ESHRE) has redefined successful IVF-ET treatment as achieving a clinical pregnancy without ovarian hyperstimulation syndrome (OHSS) that results in the birth of a single, full-term, healthy baby [2]. To achieve this outcome, clinical practice has shifted decisively toward single embryo transfer (SET), particularly single blastocyst transfer (SBT). Data from the United States Centers for Disease Control indicate a steady rise in SET utilization from 20.6% to 80.4% between 2011 and 2020; during the same period, the proportion of live births resulting from assisted reproductive technology involving multiple births declined markedly from 27.9% to 6.5% [3].
The adoption of the SBT approach has substantially reduced multiple pregnancy rates while maintaining high clinical pregnancy rates (CPRs) and live birth rates (LBRs) [4-6]. Thus, selecting the blastocyst with the strongest implantation potential for SET is essential. Morphological assessment remains a widely used, convenient, and noninvasive method for evaluating embryo viability. However, there is still no universal consensus regarding optimal blastocyst selection criteria.
A retrospective study presented at the 2024 ESHRE Congress, based on data from the German IVF registry from 2017 to 2021 and including 19,702 couples [7], compared pregnancy outcomes between couples with two embryos who chose double embryo transfer and those who underwent two SETs. Compared with double embryo transfer, a greater number of newborns resulted from two SETs (LBR: 38.5% vs. 33.1%+19.4%), with a larger proportion of these newborns being singletons (96.3%) compared with 49.5% following two-embryo transfer.
Blank et al. [8] analyzed morphological changes during blastocyst culture in fresh cycles and emphasized that embryos showing substantial improvement in quality from day 3 to day 5 were associated with better pregnancy outcomes. However, ovarian stimulation is widely known to expose the endometrium to supra-physiological estrogen (E2) levels [9], which may alter the expression of genes essential for early implantation-related endometrial remodeling [10], thereby potentially impairing receptivity and optimal implantation conditions. To improve the uterine environment and minimize these influences, selective frozen-thawed embryo transfer may offer advantages over fresh embryo transfer. Therefore, this study focused on frozen embryo transfer (FET) to examine associations between changes in morphological scores during embryonic development from day 3 to day 5 and pregnancy outcomes. The goal was to provide a stronger foundation for embryo selection to ensure that the most viable blastocyst is chosen.
Methods
1. Patients
This retrospective cohort study was conducted at two tertiary healthcare institutions: one facility enrolling patients from August 2014 to December 2022, and the other from July 2017 to December 2022. Ethical approval was obtained from the Institutional Review Boards of both institutions (approval numbers 2024-E672-01 and YX20240919-1). As data were deidentified and all analyses were retrospective, the requirement for informed consent was waived. This study was in accordance with the Declaration of Helsinki. The study population consisted of individuals who underwent SBT on day 5 during FET cycles and for whom comprehensive and satisfactory clinical data were available. Exclusion criteria included age <20 or >40 years at the time of oocyte aspiration; hydrosalpinx; adenomyosis or endometriosis; uterine abnormalities (unicornuate uterus, double uterus, saddle uterus, septate uterus, etc.); pelvic tuberculosis; uterine adhesions; endometrial thickness <7 mm on the day of transformation; changes in embryo scores before and after thawing; and donor cycles or cycles involving preimplantation genetic testing. The complete participant selection process, including all inclusion and exclusion criteria, is illustrated in Figure 1.
Patient selection and screening flowchart.
2. Controlled ovarian stimulation
A standardized protocol was used for controlled ovarian stimulation. The initial dose of gonadotropin (Gn) was determined on the basis of multiple factors, including chronological age, baseline follicle-stimulating hormone (FSH) levels, antral follicle count, and body mass index (BMI). All patients then received the appropriate Gn dose until at least one follicle reached a diameter of 18 mm or greater, at which point final oocyte maturation was triggered with either human chorionic gonadotropin (hCG) 4,000–10,000 IU or recombinant hCG (Ovidrel; Merck Serono Ltd.). For patients at risk of OHSS undergoing antagonist protocols, a GnRH agonist trigger of 0.2 mg (Diphereline; Ferring) was administered. Oocyte retrieval was performed 36 to 38 hours after triggering and was conducted under vaginal ultrasound guidance.
3. Embryo culture and assessment
Depending on semen parameters, fertilization was achieved through either IVF or intracytoplasmic sperm injection. Embryo culture was performed in accordance with standardized laboratory protocols at both centers. To ensure consistency in embryo evaluation, all assessments were conducted by two senior embryologists at each center using identical grading criteria. On day 3, embryos were evaluated following the Istanbul consensus guidelines [11], which both centers rigorously applied to minimize interobserver variability.
The decision to continue embryos to blastocyst culture was based on day 3 grading and patient preference. For blastocyst assessment, the Gardner scoring system [12] was uniformly employed, and all evaluators received standardized training to ensure consistency and reproducibility.
4. Grouping cleavage-stage embryos and blastocysts
In human embryology, a blastomere count of 7–9 on day 3 is generally considered optimal for progressing through normal developmental stages. Based on this principle and the Istanbul scoring system, day 3 cleavage-stage embryos were categorized into three groups:
(1) Excellent-D3 (n=514): 7–9 blastomeres, category I (<10% fragmentation, symmetric blastomeres).
(2) Good-D3 (n=430): 7–9 blastomeres, category II (10%–25% fragmentation, mild asymmetry).
(3) Poor-D3 (n=548): <7 or >9 blastomeres, categories I–III (>20% fragmentation, pronounced asymmetry, or developmental retardation).
Based on blastocyst morphological scores and the grouping methodology described by Lou et al. [13], day 5 blastocysts were also divided into three groups:
(1) Excellent-D5 (n=868): scores 3/4/5AA, 3/4/5AB, 3/4/5BA.
(2) Good-D5 (n=460): scores 3/4/5BB.
(3) Poor-D5 (n=164): scores 3/4/5AC, 3/4/5CA, 3/4/5BC, 3/4/5CB, 3/4/5CC.
Based on changes in morphological scores from day 3 to day 5, blastocysts were further classified into:
(1) Improvement (n=711): Poor-D3→Good-D5, Poor-D3→Excellent-D5, or Good-D3→Excellent-D5 embryos.
(2) Stability (n=568): embryos maintaining equivalent tiers (Excellent-D3→Excellent-D5, Good-D3→Good-D5, Poor-D3→Poor-D5).
(3) Deterioration (n=213): Excellent-D3→Good-D5, Excellent-D3→Poor-D5, or Good-D3→Poor-D5 embryos.
5. Endometrial preparation and vitrification
Endometrial preparation was performed using a range of protocols [14], selected according to patient preference or physician recommendation. Once the endometrium reached the target thickness (≥7 mm), vitrified blastocysts were thawed on the morning of the scheduled transfer using specialized thawing kits. Blastocyst survival was determined by re-expansion 2 hours post-warming. All thawed embryos underwent assisted hatching. With abdominal ultrasound guidance, the re-expanded blastocyst was transferred into the uterine cavity. The procedures for cryopreservation and warming, including vitrification and thawing, followed established methods previously described in detail [15].
6. Pregnancy outcomes
The primary outcomes were CPR and LBR, while the biochemical pregnancy rate and miscarriage rate served as secondary outcomes. Biochemical pregnancy, characterized by a failure to progress to clinical pregnancy, was diagnosed through a positive hCG test performed 12 days after blastocyst transfer. Clinical pregnancy was confirmed by ultrasound visualization of gestational sacs 4 to 5 weeks after transfer or by other definitive clinical indicators of pregnancy. Miscarriage was defined as a medically confirmed pregnancy loss before 28 weeks of gestation. Live birth was defined as the delivery of a viable infant at or beyond 28 weeks of gestation.
7. Statistical methods
Statistical analyses were conducted using SPSS ver. 25.0 (IBM Co.). For normally distributed data, results were presented as mean±standard deviation, and analysis of variance was used for comparisons. For non-normally distributed data, results were expressed as median (interquartile range), and the Kruskal–Wallis test (H test) was applied. Categorical variables were summarized as number (%), with group comparisons performed using chi-square or Fisher’s exact tests. Spearman’s rank correlation coefficient was used to assess correlations. To further evaluate potential influencing factors, multivariate regression analysis was performed, incorporating variables deemed clinically relevant. A p<0.05 was considered statistically significant.
Results
1. Baseline characteristics
In total, 1,492 SBT cycles were performed on day 5 after thawing. The subjects were stratified into three groups according to their morphological score progression from day 3 to day 5: the improvement group (n=711), stability group (n=568), and deterioration group (n=213). As summarized in Table 1, no significant differences were observed among these groups in age, infertility duration, insemination patterns, BMI, basal hormone levels (including FSH, luteinizing hormone, and E2), endometrial preparation protocols, endometrial thickness, or other baseline characteristics (all p>0.05).
Baseline characteristics of the different groups
2. Developmental trajectories from day 3 to day 5
Analysis of embryo-quality evolution from day 3 to day 5 showed that more than 60% of embryos classified as Excellent-D3 and Good-D3 progressed to the Excellent-D5 category. In contrast, fewer than 50% of embryos initially classified as Poor-D3 reached Excellent-D5 (Figure 2).
Trends in embryonic development at the cleavage stage.
3. Comparison of pregnancy outcomes according to day 5 grade
Given that blastocyst morphology is a key determinant of IVF success [16], it is essential to consider the implications of day 5 morphological scores in FET cycles. As presented in Table 2, the CPR and LBR of the Excellent-D5 group were comparable to those of the Good-D5 group but were significantly higher than those of the Poor-D5 group (CPR: 58.64% vs. 46.34%, p=0.014; LBR: 45.97% vs. 33.54%, p=0.012). Furthermore, an analysis across all three groups revealed no statistically significant differences in miscarriage rates or biochemical pregnancy rates.
Pregnancy outcomes of embryos transferred with different grades of blastocysts
4. Comparison of pregnancy outcomes following SET among different developmental trend groups
Table 3 provides a comparative evaluation of clinical outcomes among the improvement, stability, and deterioration groups. Compared with the improvement group, the stability and deterioration groups exhibited similar biochemical pregnancy rates (7.92% and 6.10% vs. 9.00%, p=0.384), CPR (55.28% and 55.87% vs. 57.95%, p=0.615), miscarriage rates (17.52% and 24.37% vs. 21.12%, p=0.235), and LBR (44.54% and 40.84% vs. 43.74%, p=0.648). No statistically significant differences were identified among these groups for any of the clinical outcomes (all p>0.05).
Pregnancy outcomes of single blastocyst transfer in different developmental trend groups
5. Comparison of the pregnancy outcomes of day 5 blastocysts with different day 3 morphological scores
Multivariate logistic regression analysis was used to evaluate pregnancy outcomes of day 5 blastocysts derived from embryos with distinct day 3 morphological scores (Table 4), using stabilization of quality category as the reference. All models were adjusted for infertility type, infertility duration, maternal age at oocyte retrieval, BMI, endometrial preparation protocol, and endometrial thickness.
Pregnancy outcomes of D5 blastocysts derived from different D3 morphological scores
In the Poor-D5 group, where embryos remained poor from day 3 to day 5 (reference biochemical pregnancy rate: 8.00%), embryos classified as deteriorated or improved showed comparable biochemical pregnancy rates (Good-D3: 5.71%, odds ratio [OR], 0.734, 95% confidence interval [CI], 0.135 to 3.993; Excellent-D3: 6.90%, OR, 0.818; 95% CI, 0.139 to 4.801), with no statistically significant differences (p>0.05). CPRs demonstrated nonsignificant increases for Good-D3 (51.43%; OR, 1.224) and Excellent-D3 embryos (58.62%; OR, 2.071). Live birth outcomes remained consistent across all subgroups (31.03%–37.14% vs. 33% reference).
For Good-D5 blastocysts (reference biochemical pregnancy rate, 8.33%), biochemical pregnancy rates showed only minimal variation by initial morphology (Poor-D3: 9.50%, OR, 1.314; Excellent-D3: 6.04%, OR, 0.731). CPRs were modestly higher in Poor-D3 embryos (58.66% vs. 53.79% reference; OR, 1.181) and comparable in Excellent-D3 embryos (56.38%; OR, 1.081). LBRs were similarly consistent across all subgroups (42.42% to 43.62%).
The most clinically meaningful results appeared in the Excellent-D5 group. Good-D3 embryos progressing to Excellent-D5 demonstrated outcomes equivalent to the reference group across all indicators. In contrast, Poor-D3 embryos upgraded to Excellent-D5 showed significantly lower LBR (40.52% vs. 48.81%: OR, 0.714; 95% CI, 0.513 to 0.994; p=0.046). CPRs were also slightly lower (54.28% vs. 60.12%; OR, 0.785), although this difference did not reach statistical significance.
Discussion
Despite ongoing debate regarding the predictive value of day 3 embryo morphology for blastocyst transfer outcomes, low cell number, high fragmentation, and asymmetry are known to correlate with reduced implantation rates [17,18]. However, outcomes in fresh embryo transfer cycles are confounded by ovarian stimulation–induced supra-physiological E2, which can impair endometrial receptivity [9,10]. To isolate the effects of embryo morphology alone, we conducted a dual-center retrospective study of 1,492 day 5 frozen-thawed SBTs.
Consistent with the established consensus, D5 blastocyst morphology strongly predicted pregnancy success [13,19,20]. Excellent-D5 blastocysts outperformed Poor-D5 counterparts in CPR (58.64% vs. 46.34%) and LBR (45.97% vs. 33.54%), while maintaining comparable biochemical pregnancy and miscarriage rates. These findings are aligned with evidence showing lower aneuploidy rates in high-grade blastocysts [21-23] and the selective secretion of hsa-miR-320a by high-quality preimplantation embryos. This microRNA promotes human endometrial stromal cell migration, thereby contributing to a receptive endometrial microenvironment that facilitates implantation [24]. Day 3 embryo quality also correlated with blastocyst formation rates (65.37% of Excellent-D5 originating from Excellent-D3 vs. 49.08% from Poor-D3), supporting the superior early developmental potential of high-grade D3 embryos [25-27], while confirming that poor-quality D3 embryos may undergo ‘self-correction’ to form viable blastocysts [28].
The core clinical question—whether morphologically comparable D5 blastocysts differ in outcomes based on D3 origin—was evaluated using trajectory-specific and stratified analyses. Across all D5 quality grades, CPR (55.28%–57.95%, p=0.615) and LBR (40.84%–44.54%, p=0.648) did not differ significantly among the improvement, stability, and deterioration groups. These results contrast with the findings of Blank et al. [8], whose fresh-cycle data were influenced by supra-physiological E2 exposure and relied on oversimplified trajectory classifications. Stratified analyses further demonstrated minimal variation in outcomes attributable to D3 origin among Poor-D5 and Good-D5 blastocysts, with no significant differences in CPR or LBR.
Notably, within the Excellent-D5 cohort—the key group for clinical refinement—Poor-D3-derived blastocysts exhibited slightly lower CPR (54.28% vs. 60.12%; OR, 0.785) and a higher miscarriage rate (21.92% vs. 16.83%; OR, 1.34) compared with Excellent-D3-derived blastocysts. These differences culminated in a significantly reduced LBR (40.52% vs. 48.81%; OR, 0.714; 95% CI, 0.513 to 0.994; p=0.046). This pattern contrasts with the findings of Bartolacci et al. [29], likely due to inconsistent morphological grading criteria across studies. Although statistically significant, this result should be interpreted with caution, as the subgroup sample size may limit the robustness of the observed effect.
Collectively, our findings support two critical clinical messages: (1) day 3 morphology has limited predictive value for overall D5 blastocyst outcomes, reinforcing the practice of extending culture of suboptimal D3 embryos rather than discarding them prematurely; and (2) day 3 origin provides prognostic refinement within top-tier D5 blastocysts, as Poor-D3-derived Excellent-D5 embryos show moderately reduced live birth potential, potentially related to persistent chromosomal mosaicism [30]. These results support a dual-axis evaluative framework that integrates D5 morphology as the primary determinant and developmental history as a secondary modifier to optimize embryo selection in FET cycles.
The study’s limitations include its retrospective design (partially mitigated by stringent inclusion criteria), inherent subjectivity of morphological grading, and the absence of preimplantation genetic testing data. Additionally, caution is warranted when interpreting the significant finding within the Excellent-D5 subgroup, as smaller subgroup sizes may affect the stability of this comparison. Future prospective, multicenter studies incorporating objective assessment tools (e.g., time-lapse imaging, artificial intelligence) are needed to validate and extend these findings.
Notes
Conflict of interest
No potential conflict of interest relevant to this article was reported.
Acknowledgments
The authors wish to thank all staff at both centers for their help with data collection. The data presented in this study are available from the corresponding author upon reasonable request.
Author contributions
Conceptualization: QG, WM, LL, BL, YY. Methodology: QG, QH. Formal analysis: QG, LL, BL. Data curation: QG, LL, FL. Funding acquisition: QH, YY. Software: LQ, LR. Validation: ZD, CY. Investigation: WM, FL. Writing-original draft: QG, WM, LL. Writing-review & editing: QG, QH, FL, LQ, LR, ZD, CY, BL, YY. Approval of final manuscript: QG, WM, LL, QH, FL, LQ, LR, ZD, CY, BL, YY.