Since the introduction of in vitro fertilization (IVF) in 1978, advances in embryo selection strategies have played a pivotal role in improving clinical outcomes and reducing the incidence of multiple gestations through the implementation of single embryo transfer (SET) [1]. Traditionally, blastocyst selection has depended on static morphological assessment, particularly the Gardner and Schoolcraft grading system, which evaluates blastocyst expansion, inner cell mass (ICM), and trophectoderm (TE) quality [2]. Although widely adopted, these qualitative evaluations are inherently limited by subjectivity and interobserver variability, and they may not fully reflect the developmental potential of an embryo.

In recent years, the application of quantitative morphometric parameters has attracted increasing attention as a potentially more objective approach to embryo selection. Metrics such as ICM area and estimated TE cell count have shown strong associations with ongoing pregnancy outcomes, whereas blastocyst diameter alone has demonstrated inconsistent predictive value [3]. These findings suggest that reproducible and measurable features of blastocyst morphology may provide additional insights beyond traditional grading systems.

Moreover, conventional morphological variables—including TE and ICM grades, expansion stage, and the day of blastulation (e.g., day 5 vs. day 6 or 7)—have been independently linked to live birth rates in frozen embryo transfer (FET) cycles [4,5]. A recent large-scale study involving more than 10,000 FET cycles identified ICM grade as the strongest morphological predictor of both live birth and miscarriage risk [6].

Despite this evidence, relatively few studies have explored the combined predictive value of morphometric and morphological parameters, particularly in frozen single blastocyst transfer. Most previous work has either examined fresh transfers or analyzed these parameters in isolation, limiting relevance to current practice in which FETs predominate.

Therefore, this retrospective study seeks to evaluate the relationship between selected morphometric parameters—including blastocyst diameter—and clinical outcomes in frozen single blastocyst transfer cycles. By integrating these quantitative measures with conventional Gardner and Schoolcraft morphological grading, we aim to identify predictive combinations that may enhance the accuracy of embryo selection, improve SET outcomes, and advance standardization in embryology laboratories.

A retrospective analysis was conducted with data collected from patients undergoing frozen SET cycles from 2021 to 2024. Informed consent form was obtained from all patients (Study no. 37/ECR/1312/Inst/TG/2019/RR-24). Inclusion criteria: This analysis included data from self-patients who have undergone single vitrified-warmed blastocyst transfers. Exclusion criteria: Cycles involving the transfer of cleavage-stage embryos, embryos genetically tested before transfer, and all cycles within a donor program (e.g., embryos derived from donor oocytes or donor embryos) were excluded. All blastocysts were morphologically graded using the Gardner grading system. Based on blastocoel expansion level and hatching status, embryos were numerically graded from level 1 to 6. ICM and TE quality were graded as A, B, or C depending on cell number and arrangement, as follows: ICM quality (grade A: many cells tightly packed; grade B: several cells loosely packed; and grade C: few cells); TE quality (grade A: many cells forming a cohesive epithelium; grade B: few cells forming a loose epithelium; and grade C: very few large cells).

In addition, the degree of expansion was assessed morphometrically by measuring the inner diameter of the blastocyst from images taken at the equatorial plane during embryo transfer (ET) using Hamilton Thorne laser software (Hamilton Thorne Inc.). Multiple statistical tests were performed to analyze both qualitative and quantitative assessments of blastocysts in relation to clinical outcomes.

A total of 170 SET cycles were performed during the study period. After excluding donor programs and pre-implantation genetic testing for aneuploidy (PGT-A) embryos, 157 FET cycles that met the inclusion criteria were analyzed (Figure 1). The sample size was sufficient to enable robust group comparisons and to allow meaningful interpretation of the associations between blastocyst grading and reproductive outcomes. The mean female age of the study population was 32.37±4.29 years, which was similar across both the pregnancy-positive and pregnancy-negative groups. Patient characteristics are summarized in Table 1.

When comparing each morphological and morphometric parameter of the blastocyst with clinical outcomes, both ICM grade and TE grade were found to significantly influence pregnancy outcome, IR, and CPR (Table 2).

Blastocysts with Grade A ICM and TE exhibited significantly higher clinical outcomes compared with grades B and C. By contrast, blastocysts with grade C ICM or TE quality demonstrated markedly poor pregnancy outcomes. Although the mean diameter of blastocyst expansion was higher in the pregnancy-positive group (279.25±2.16 µm) compared with the negative group (132.19±2.19 µm), this difference did not reach statistical significance. Morphological assessment showed that blastocysts with level 2 expansion had the lowest clinical outcomes (TPR: 20%; IR: 0%; CPR: 0%). Expansion levels 3 (TPR: 54.2%; IR: 54.2%; CPR: 54.2%) and 4 (TPR: 52.13%; IR: 50%; CPR: 46%) demonstrated comparable outcomes, but without statistical significance. As per the laboratory’s cryopreservation protocol, only blastocysts with expansion greater than level 2 were vitrified; thus, no level 1 blastocysts were available in the post-thaw cohort. All FETs were performed 2–4 hours after thawing, following adequate re-expansion of the blastocyst. At the time of transfer, none of the blastocysts displayed hatching or TE herniation.

Overall, multivariate analysis revealed that TE grade and morphological assessment of blastocyst expansion level had significant effects on total pregnancy outcome, IR, and CPR (Table 3). None of the parameters showed significance with miscarriage rate, likely due to the limited data available.

The success of IVF treatment is strongly influenced by the selection of high-quality embryos for uterine transfer. Advances in culture systems and laboratory techniques have increased the use of human blastocyst culture, enabling improved embryo selection for transfer. At present, blastocyst assessment primarily relies on subjective morphological evaluation. Incorporating morphological and morphometric analyses could reduce observer variability [9]. However, there remains a paucity of research on automated image analysis of human embryos [10-12], particularly at the blastocyst stage [13,14]. Automated approaches such as time-lapse monitoring and PGT-A offer potential alternatives to traditional morphology-based scoring. Yet, both approaches face technical and biological limitations, in addition to being costly. While PGT-A can provide IRs largely independent of blastocyst morphology once euploid embryos are selected, several studies indicate that morphologically high-quality blastocysts are more likely to be euploid compared to lower-quality ones [15-17]. Thus, although PGT-A offers an advanced genetic assessment that refines embryo selection, conventional morphology remains a valuable and more accessible method, particularly in centers without PGT-A facilities or for patients unable to afford such testing. The present study aimed to identify specific blastocyst characteristics predictive of clinical implantation following FET by evaluating multiple morphological and morphometric parameters, including expansion score, ICM quality, TE quality, and mean blastocyst diameter. Our findings demonstrated that higher qualitative expansion grades and superior TE quality were significantly associated with improved clinical outcomes.

The TE, a key component of the blastocyst, plays a critical role in establishing and maintaining a successful pregnancy. During the peri-implantation period following ET, TE is engaged in several essential processes for uterine receptivity and fetal development. These include hCG-mediated signaling, hatching from the zona pellucida, adhesion to and invasion of the endometrium, and modulation of the maternal immune response [18-22]. The collective importance of these TE-mediated processes highlights their central role in implantation and early embryonic development. The predictive value of TE morphology observed in this study is consistent with this understanding, supporting the idea that TE structural and functional integrity provides valuable insight into implantation potential.

Several studies have reported that blastocyst expansion is an independent predictor of live birth rates in fresh single blastocyst transfer cycles [23,24]. Du et al. [25] further demonstrated that blastocoele expansion degree may better predict live birth outcomes than ICM or TE grades, in both fresh and vitrified/warmed single blastocyst transfers. Expansion is intrinsically linked to the TE, as cohesive TE cells restrict blastocyst fluid outflow while actively pumping sodium ions into the cavity. This osmotic regulation drives water influx and facilitates blastocoele expansion. Thus, a fully expanded blastocyst reflects functional TE cells, with this functionality more dependent on cellular molecular quality than on absolute cell number or arrangement. In line with these findings, although the positive group in our study had a larger mean blastocyst diameter, this metric was not statistically significant for predicting outcomes. In contrast, the morphological assessment of expansion stage significantly correlated with clinical outcomes such as pregnancy and IRs.

Although previous studies have consistently shown that ICM quality correlates with implantation potential, our study did not detect a statistically significant association between ICM grading and implantation (Table 2). This discrepancy is likely attributable to the relatively small sample size in our cohort, which limited the statistical power to detect differences [26-28].

In conclusion, our study demonstrated that in the selection of frozen blastocysts for transfer, priority should be given to morphological assessment of blastocyst expansion level and TE quality, as these parameters play a crucial role in predicting clinical success following frozen-thawed ET. Notably, blastocysts with the highest TE quality achieved pregnancy and IRs of 64.7%. The observed discrepancy between qualitative and quantitative expansion underscores the multifaceted nature of blastocyst expansion, suggesting that success is determined not solely by size, but also by intrinsic cellular functionality and dynamic developmental processes.

A key strength of this study lies in its direct comparison between traditional qualitative methods (Gardner grading) and quantitative assessment (blastocyst diameter measurement), thereby addressing the important need for more objective embryo selection criteria.

However, this study has limitations. Being retrospective in design and based on a relatively small sample size of frozen-thawed blastocysts, its findings should be interpreted with caution. Furthermore, live birth outcomes can be influenced by numerous factors beyond blastocyst quality. Larger datasets and prospective studies are therefore needed to validate these findings.

The findings suggest that simple diameter measurements alone are insufficient for robust prediction of clinical outcomes. Future research should explore more advanced morphometric parameters for ICM and TE, including volume, cell number, and cellular arrangement, integrated with artificial intelligence and machine learning approaches. Such developments could support the creation and refinement of artificial intelligence-based embryo selection tools, ultimately advancing standardization and precision in IVF practice.