Research Question: Is embryo development rate associated with a higher rate of euploidy.

Design: Retrospective observational study.

Results: Of the 1619 biopsied and PGT-A tested embryos, 880 (54.4%) were day 5 blastocysts and 714 (44.1%) reached the blastocyst stage on day 6. The remaining 25 (1.5%) embryos were expanded blastocysts on day 7. The proportion of euploid embryos among day 5 blastocysts (50.8%) was significantly greater (p<0.0001) than that of day 6 blastocysts (35.9%). Corresponding aneuploidy rates were lower (p<0.0001) on day 5 (32.0%) compared to day 6 (44.0%).

Conclusion: Our results suggest that the genetic composition of an embryo is associated with the rate of embryo development, with day 5 blastocysts demonstrating a significantly higher likelihood of being euploid. This information is clinically important and provides valuable, non-invasive criteria regarding embryo quality, which can be used in embryo selection to improve clinical outcomes in non-PGT-A patients.

• PGT-A; Morphokinetics; Biopsy; Euploidy; Blastocyst

Amin N, Willoughby K, Moffatt L, Neal T, Tovar M, et al. (2026) Euploidy Status is Correlated with Day of Blastocyst Formation. J Embryol Develop Biol. 5(1): 1008.

Blastocyst formation results from many sequential steps that can be used to track embryo development. First, the zygote cleaves into many blastomeres, which eventually compact into a morula. As the outer cells differentiate into trophectoderm (TE), they cavitate, allowing fluid to enter the morula creating the liquid blastocoel. The TE cells begin to pump sodium into the cellular body, creating a concentration gradient across the membrane. As the morula continues to expand through osmosis, a fully expanded blastocyst is formed. The rate and order in which these steps occur are an important measure for the normal development of human embryos and can ultimately determine if an implanted embryo results in a live birth.

The field of assistive reproduction focuses on extracting oocytes and combining them with spermatozoa through conventional in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) to form embryos that would otherwise not be naturally conceived. First originating as early as ancient Egyptians incubating chicken eggs in ovens, the prospect of using external means to aid reproduction has been consistently thought over through most of human history [1]. Ever since the first successful birth from artificial implementation in 1978 [2,3], variations of this practice along with IVF have become routine practice to aid many forms of infertility [4]. In addition, the more widespread usage of digital databases as well as emphasis on lab training have been crucial to the education and rising commercial usage of these practices [5]. Most recently, the use of artificial intelligence and machine learning to assist in PGT-A test results has been able to predict outcomes with a comprehensive accuracy of around 83% [6]. This work has the potential to also help extrapolate some of the unknown potential causes for biochemical pregnancy loss [6]. As research continues to rapidly expand, the best and most appropriate guidelines are always changing and selection criteria for embryo transfer to provide the best chance of pregnancy is an ongoing challenge for embryologists around the world.

Developmental milestones such as time of syngamy and early cleavage has been suggested as a reliable predictor of implantation when selecting embryos for transfer on day 3 [7,8]. Early time-lapse imaging studies revealed blastocysts that resulted from completed first and second cell divisions within 25.90 and 37.88 hours in culture produced higher pregnancy rates [9]. Racowksi et al. [10], reported that the number of embryos at the 8-cell stage in a cohort is a predictor of blastocyst formation. More recently time to blastulation among donor eggs was shown to be correlated with implantation and live-birth outcomes [11]. Some have hypothesized that spindle orientation within the zygote can more heavily influence chromosomal status than once initially thought [12,13], whereas others have shown that the quantity and distribution of mitochondria in early stage embryonic cells can greatly affect the organization of chromosomes, resulting in aneuploidy [14].

Significant focus has been placed on grading the inner cell mass (ICM) and TE as well as expansion of blastocysts. Over the years there has been ample conflicting data as to the most important variable. Lee et al., looked at morphokinetics and showed that aneuploid embryos took longer to develop into blastocysts [15]. It seems reasonable to presume that since rate of blastulation has been correlated with live birth, and euploidy is the strongest predictor of live birth, blastocysts that develop by day 5 are more likely euploid.

Given the complexity and importance of the blastocyst formation process, accurate assessment of development is crucial to improving future reproductive outcomes. Despite the large amount of advancements regarding time-lapse imaging (TLI) technology, the debate to determine the most reliable indicators of viability continues today. This study aims to address some gaps in this area by concentrating on the correlation between euploidy and timing of blastocyst formation. Our data allows us to refine and improve current embryo selection processes that can potentially raise live-birth rate probabilities with very minimal cost and ease of implementation.

Patient Population

This retrospective observational study included 1619 autologous embryos biopsied from 403 patients between age 25 and 44 who underwent IVF with ICSI and PGT-A from Jan 1, 2022 to July 15, 2024 at ONE Fertility (Burlington, Ontario, Canada). Patients were given either a gonadotropin releasing hormone antagonist or agonist controlled ovarian stimulation cycle. After appropriate follicle growth and rise in estradiol levels patients were triggered with human chorionic gonadotrophin and oocytes were collected approximately 36 hours later. Semen samples were provided by the male partner and processed by a standard swim-up procedure. Cumulus oocyte complexes (COCs) were isolated from follicular fluid collected by transvaginal ultrasound guided needle (Cook,Sydney, Australia) aspiration. ICSI was performed 3-4 hours after oocyte retrieval on oocytes that had achieved nuclear maturation as evidenced by the presence of the extruded first polar body. COCs were denuded by being exposed briefly (15-20 seconds) to hyaluronidase (Origio, Denmark) and gently aspirated several times to remove residual granulosa cells. Sperm was selected for ICSI based on morphology and progressive motility using an Axiovert 200 microscope (Zies, Germany) equipped with a Narishige micromanipulation system (Tokyo, Japan).

Biopsy and PGT-A

Zygotes derived from IVF/ICSI were cultured in a single step continuous media (Global Total - CooperSurgical®, USA), channelled (Saturn V laser - CooperSurgical®, USA) on day 3 of development and cultured to the blastocyst stage as per standard IVF/ICSI/PGT procedures. On day 5 and/or 6 and/or 7, TE cells herniating through the channel in the zona pellucida were biopsied with the aid of a Saturn V laser (CooperSurgical®, USA). Biopsied cells were rinsed through PBS and placed in RNA-free Eppendorf tubes with 0.2 ml of PBS (CooperSurgical®, USA) for genetic analysis by next generation sequencing (NGS - Cooper Genomics, USA). The genetic report of the biopsied cells diagnosed embryos as euploid, aneuploid, mosaic (high or low level) was used to stratify the embryos for this study. Some cases had no result due to insufficient quality DNA for testing.

Statistical Analysis

Variables are expressed as means ± standard deviation. A one-way ANOVA was used to compare differences between the three groups stratified by age. Chi-square analysis was conducted to determine differences between the day 5 and day 6 outcomes. A p-value <0.05 considered statistically significant.

Of the 1619 biopsied and PGT-A tested embryos, 880 (54.4%) were day 5 blastocysts and 714 (44.1%) reached the blastocyst stage on day 6. The remaining 25 (1.5%) embryos were expanded blastocysts and biopsied on day 7. Among these blastocysts, 7 were euploid, 15 were aneuploid, 1 was a high-level mosaic, 1 was a low-level mosaic and 1 did not produce a result. Day 7 blastocysts were excluded from further statistical analysis as a result of the small sample size. The mean patient age was 35.5 ± 3.7 and the average number of embryos biopsied was 4.0 per patient overall. Baseline patient characteristics and cycle outcomes are summarized in Table 1 and 2 respectively.

Table 1: Baseline patient characteristics.

*ARA=advanced reproductive age; DOR=diminished ovarian reserve

Table 2: Oocyte characteristics and embryo development outcomes stratified by female age.

* one way ANOVA comparing age groups ≤ 35, 35 to < 40, and ≥ 40 with a p-value < .05 considered statistically different.

** Tukey post hoc analysis to facilitate pairwise comparisons within the ANOVA table with a p-value < .05 considered statistically different.

Despite a greater number of oocytes retrieved and mature oocytes for patients in the ≤ 35 and ≥ 40 age groups compared to those in the 35 to < 40 age range there was no difference in the oocyte maturation, fertilization and blastocyst rate (Table 2). Patients in the younger group (≤ 35) had a significantly higher (p < 0.001) number of embryos reaching blastocyst on day 5. There was no difference in the number of blastocysts formed on day 6 across all age groups (Table 2).

The proportion of euploid embryos among day 5 blastocysts (50.8 %) was significantly greater (p < .0001) than that of day 6 blastocysts (35.9 %). Corresponding aneuploidy rates were lower (p < .0001) on day 5 (32.0%) compared to day 6 (44.0%). There were more embryos diagnosed as Low Level Mosaics (LLM, p = .002) on day 6 compared with their counterparts on day 5. There was not any difference between High Level Mosaics (HLM, p = .523); or embryos with a no result report (p = .825) observed (Table 3).

Table 3: Distribution of PGT results from all ages stratified by day of blastocyst formation and biopsy for PGT.

* chi-square comparison between blastocyst development on day 5 vs day 6 with a p-value < .05 considered statistically different.

When stratified by age, there were significantly more euploid embryos in the group of patients ≤ 35 (p < .0001) and between 35 to < 40 (p = 0.042). Among patients ≥ 40 there were slightly more euploid embryos on day 6 compared to day 5 although not statistically significant (Figure 1).

Figure 1: Percentage of euploid blastocysts stratified by day of blastulation/biopsy for PGT-A, *P<0.0001;** p=0.042; and NS-not significant (p=0.426).

A significant difference in euploidy rates was observed between day 5 (50.8%) and day 6 (35.9%) blastocysts. This aligns with our hypothesis that embryos reaching the blastocyst stage on day 5 have a much higher likelihood of being chromosomally normal. Similarly, the escalation of aneuploidy rates within the day 6 embryos suggest that slower embryo development could be an indicator of chromosomal abnormalities. It is also interesting that this trend was consistent despite maternal age, albeit with decreasing impact. For women over the age of 40, day 6 blastocyst showed a slightly higher probability of being euploid (27.2%) than day 5 blastocysts (24.1%). This deviation could be attributed to the smaller sample size of the older age group. However, the trend of earlier developing blastocysts having higher euploidy rates is prominent within the younger patients [16,17]. This outcome could potentially be explained through ARA/DOR also causing delays in blastulation independent of euploidy status or chromosomal development. In the context of an older age group, suboptimal development of a blastocyst may not necessarily dictate a chromosomally affected embryo [17]. The timing of blastulation may serve as a different indicator for aneuploidy among younger women, whereas for older patients, it might reflect regular development patterns. Aside from age, the cause of infertility didn’t elicit a great impact on the development of blastocysts or embryo euploidy. Due to the non-linear correlation of these components, as well as the complex nature of embryo development, it is likely that there are still other factors at play [18]. Currently, the morphological grading system which includes blastocoel expansion, quality of the ICM, and TE quality remains a critical resource for embryo selection [19]. When choosing between embryos of the same ranking, those that have blastulated on day 5 should be given higher priority over similar embryos on day 6 [20]. It is possible that aneuploidy as well as timing of blastulation are not completely independent of these factors, and that an embryo’s overall quality may affect its implantation rate.

Growth rate has proven to be a significant factor in the evaluation of embryo quality. With the expansion of newer TLI technology, this has remained true as cell numbers as well as other developmental behaviors can be assessed more precisely. Some previous studies utilising TLI suggested that cell number of the embryo throughout the development process was a large indicator of whether or not a blastocyst was formed. In particular, embryos with seven or more cells on day three of development tended to create significantly more good quality and regular quality blastocysts [21]. However, it was also noted that 85.7% of the embryos containing 7-8 cells on day three exhibited normal division behaviour, whereas only 45.8% of embryos containing 9-10 cells on day three exhibited normal behaviour [21]. Various clinical trials have also observed that the live birth rate (LBR) is linked to the timing of blastulation [22]. In one study, the LBR of excellent day 5 and day 6 embryos respectively were 62.14% and 53.61%, and the LBR of good day 5 and day 6 embryos were 45.18% and 32.21% respectively. In both good and excellent embryos, those that blastulated on day 5 were significantly more viable than their day 6 counterparts [22,23]. Similarly, embryos with faster growth speeds tend to have higher implantation rates than slower growing embryos [23]. Though with relatively small sample sizes, it has also been deduced that lower mitochondrial content in the later stages of development has resulted in higher euploidy rates [24]. Assessed through NGS, the ratio between mitochondrial and nuclear DNA was observed to be the same on day 3 of development, regardless of euploidy status; yet by day 6 of development, this ratio decreased dramatically within euploid embryos [24-26]. In addition, it was also observed that within euploid blastocysts, smaller amounts of mitochondrial content resulted in faster developing blastocysts, and that fully expanded blastocysts generally had less mitochondrial content that early-stage blastocysts. These developments may contribute to the small amounts of evidence showcasing more success in blastocyst-stage transfer over cleavage stage transfer [27]. It has also been thought that the number of TE cells was the singular greatest indicator for embryo viability [28]. However, as more recent research has shown, the timing of blastocoel development has become increasingly prevalent [23]. As previously stated, the current morphological grading system places higher priority on blastocoel expansion and growth status, however timing of blastulation as a factor has only been recently explored. Because of this lapse in information, it would be best to continue exploring these factors and how they affect euploidy and live birth rates.

Another study revealed a link between tobacco usage and delayed blastulation. Utilising a group of women and men with similar ages and BMIs, as well as relatively unvarying reasons for infertility, it was noted that the ratio between active smokers and non-smokers was significantly larger within day 6 embryos than day 5 (13.6% vs 8.7%) [29]. Similarly, women who actively smoked often went through more assisted reproductive technology cycles than others. Though the exact cause for this correlation remained unexplored, it is possible that the tobacco usage impacted the euploidy and developmental rate of the embryos. Similarly, there is substantial evidence to showcase that Vitamin D levels within the mother seemed to correlate with significantly more euploid blastocysts [30]. Though it still remains unclear if this issue can be combated by supplements [31], it was observed that after controlling for age, BMI and other factors, small increases in Vitamin D could substantially increase the odds of producing a euploid embryo. Alternatively, whether or not paternal age relates to blastulation timing is another detrimental factor that's currently being explored [32]. It has been previously noted that for women involved in controlled ovarian stimulation resulting in live birth, each increase of year in paternal age generally resulted in a 6% increased probability that blastulation would occur on day 6 [33]. However, when utilizing frozen-thawed embryo transfer, no statistical significance was observed [33]. Though paternal age can serve as a factor to aneuploidy in some cases, it has been shown to have little impact between smaller time intervals, and often negligible overall.

Strengths and Limitations

This study was completed using a relatively large sample-size of embryos. As a result, the trends that have been observed are more likely to be significant. Similarly, implementation of these findings would not be difficult as a result of its simplicity. Without the need for additional technology, the timing of blastulation can be utilized as a more prominent selection factor in clinics worldwide.

Unfortunately, this study wasn’t able to account for variables such as tobacco usage, or vitamin D deficiency that have previously been shown to likely impact euploidy. Additionally, our sample sizing in the subsection of women over the age of 40 was relatively small, possibly resulting in a skewed outcome. To increase this specific subsection, it may have been beneficial to include multiple centres, however, controlling variables across multiple clinics may be difficult, resulting in some biases.

The rising importance of blastulation timing could indicate a need for a more nuanced approach when choosing which embryos to transfer. This evolving perspective also raises critical questions of how and when it should be prioritized. For instance, some cases have shown that timing of blastulation only makes a significant difference in women under the age of 35 [16], whereas others have shown that the freezing method of the blastocyst can mitigate the effects of this timing entirely [34]. Even so, chromosomal mutation within the meiosis and mitosis stages of development still appear to play a role in delayed blastocyst development [35]. For patients undergoing PGT-A testing, these findings could influence their decisions regarding treatment, as well as the advice received from physicians and other medical experts. If transfer at the blastocyst stage instead of at the cleavage stage begins to yield more positive results, patients may opt to delay the implementation [36]. Additionally, the development of blastocysts could eventually influence the decision on whether or not a patient should receive PGT-A testing, and its necessity. As research progresses, the status of an embryo achieving blastocyst stage on day 5 and hatching stage on day 6 may start to be seen as a prognostic factor of IVF treatment [36]. Understanding these correlations and integrating morphokinetic evaluations into clinical practice could enhance outcomes not just for PGT-A patients but also for those not undergoing genetic testing [37]. Despite conflicting results seen within blastocysts after undergoing frozen transfer, the impact of their rate of development may affect the method of freezing used [38]. Within slow-freezing, day 5 and day 6 embryos showcased no significant difference in euploidy, however, following vitrification, timing of blastulation remains important post-transfer [38]. Depending on the equipment available, this may further influence both the patients and physician’s opinion on the relevance of an earlier developed blastocyst. Taken together, the correlation between timing of blastocyst formation in our study and supported by others provides a cost-effective and efficient criterion to rank embryos for transfer.

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