Introduction
During nuclear and cytoplasmic maturation, oocytes accumulate and systematically organize cytoplasmic components such as organelles, inter-metabolites, and cytoplasmic determinants [1-3]. Fertilization triggers a significant cytoplasmic transition, establishing zygotic competency through the formation of structures like mitochondria, lysosomes, and cortical granules. This pivotal event initiates a complex sequence of cellular and molecular changes, including the central migration of the pronucleus, the fusion of parental genomes, and the transition from meiosis to mitosis, which are essential for the development of new organisms [4,5]. During this period, the fertilized oocyte attains totipotency at molecular and cellular levels [6,7]. Consequently, it is not surprising that the developmental trajectory of the embryo is closely linked to the events of fertilization [8,9].
The prediction of developmental potency in oocytes and fertilized oocytes is crucial in assisted reproductive technology (ART). Efforts have been made to identify markers that indicate the competency of oocytes and fertilized embryos. Traditionally, morphological grading of embryos or zygotes has been used to select candidates for embryo transfer. However, this method alone does not guarantee successful full-term development. Conversely, quantitative analysis of chromosomes has been explored using various methods in ART, as aneuploidy is a known cause of spontaneous miscarriage [10,11]. Although studies have examined the correlation between zygote morphology and embryo competence, the clinical efficacy of these findings remains a subject of debate [12,13]. Over the past several decades, research on the various events occurring during fertilization in in vitro fertilization (IVF) has been limited. However, the introduction of time-lapse (TL) technology has yielded valuable insights into many critical fertilization processes. Pronuclei and nucleoli have been utilized as criteria for cryopreservation and assessing chromosomal copy number [14,15]. Additionally, the combination of pronuclear and blastocyst morphology has been used to predict embryo ploidy by assessing age [16]. Utilizing TL technology, Cavazza et al. [17] suggested a link between nucleolar clustering and the presence of healthy euploid embryos in human zygotes. However, the criteria proposed so far are not entirely sufficient for identifying competent embryos.
To advance our understanding of the clustering kinetics of nucleolar precursor bodies (NPBs) in zygotes and explore their potential application in assessing embryo competence, we investigated the relationship between NPB clustering and morphokinetics during preimplantation embryo development. Additionally, we examined the incidence of aneuploidy in preimplantation genetic testing for aneuploidy (PGT-A) cycles.
Methods
1. Study population
This study analyzed data from 200 blastocysts obtained during 53 PGT-A cycles at the Maria Fertility Center between January 2023 and December 2023. These cycles included patients who had experienced recurrent implantation failure (RIF) or recurrent pregnancy loss (RPL). RIF was defined as the failure to observe a gestational sac on ultrasound after at least three fresh or frozen cycles. RPL was characterized by more than two miscarriages before reaching 20 weeks of gestation. The exclusion criteria for the study were parental chromosomal abnormalities. The diagnosis of infertility encompassed various factors, including male factors, female factors, or a combination of both (Table 1). The Institutional Review Board (IRB) of Maria Fertility Hospital approved this study (IRB reference number: HR-2023-46-01). Written informed consent by the patients was waived due to a retrospective nature of our study.
2. Oocyte retrieval and intracytoplasmic sperm injection
Ovarian stimulation was achieved using a combination of long and short gonadotropin-releasing hormone agonists (Decapeptyl, Ferring Pharmaceuticals; or Lorelin Depot, Dongkook Pharm) and antagonists (Cetrotide, Merck-Serono; or Orgalutran, Organon), supplemented with human menopausal gonadotropin (IVF-M HP, LG Chem; or Menopur, Ferring Pharmaceuticals). The dosage of gonadotropins was tailored based on the follicular response, monitored through transvaginal ultrasonography. When two or more follicles reached 17 or 18 mm in diameter, a subcutaneous injection of 250 µg of recombinant human chorionic gonadotropin (hCG) (Ovidrel; Merck-Serono) was administered. Oocyte retrieval occurred 35 to 36 hours post-hCG injection. Oocyte denudation involved the use of Maria Research Center (MRC)#wash media (Maria Medical Foundation) containing 0.1% hyaluronidase. Intracytoplasmic sperm injection (ICSI) was carried out using MRC#ICSI media (Maria Medical Foundation). After ICSI, oocytes were individually cultured in oil-covered, pre-equilibrated MRC#13 medium (Maria Medical Foundation) within EmbryoSlide dishes (Vitrolife). Culturing was conducted in a TL incubation system, the EmbryoScope+ (Vitrolife).
3. Embryo culture and time-lapse recording
Fertilization was assessed 15 to 18 hours after ICSI by the presence of a pronucleus and initially cultured in MRC#13 until day 3. Subsequently, the culture was transferred to MRC#46 (Maria Medical Foundation) until reaching the blastocyst-stage (day 5 or 6 post-ICSI) at EmbryoScope+. The culture conditions maintained were 37 °C, 6.0% CO2, 5.0% O2, and balanced N2. Morphokinetic parameters were recorded, as detailed in Table 2.
4. Observation of nucleolar precursor body clustering
TL images of zygotes were captured every 10 minutes across seven focal planes on the Z-axis (15 μm) using EmbryoScope+ and EmbryoViewer software (Vitrolife). The morphokinetics of NPBs were annotated by three senior investigators. The kinetics of NPB clustering were analyzed across all Z-plane images from the time of pronuclear formation until pronuclear breakdown (PNBD). Clustering of NPBs was determined using the last frame before PNBD (approximately 24±18 minutes before PNBD): the boundaries of the pronuclei and NPBs, as well as the interface of the two pronuclei, were manually annotated using the support tools in EmbryoViewer. Zygotes were classified as clustered (CL) when all NPBs were located within half the distance from the pronuclei interface. Conversely, zygotes were classified as non-clustered (NCL) when more than one NPB was located beyond half the distance from the pronuclei interface (Figure 1).
5. Biopsy and PGT-A
For the zona pellucida (ZP) prehatching protocol, a 5 μm hole was created in the ZP using a laser on day 3. Only blastocysts that were hatching or fully hatched, with a clearly defined inner cell mass and a cohesive trophectoderm (TE) epithelium consisting of multiple cells, were selected for biopsy. On the biopsy day, 5 to 10 TE cells were collected by flicking. The biopsied TE cells were then washed in 1×phosphate buffered saline and transferred into a polymerase chain reaction tube. These cells were analyzed using commercial genetic testing tools (array comparative genomic hybridization, aCGH; DxVx). The analysis began with complete genome amplification, followed by aCGH to examine the biopsied cells.
6. Statistical analysis
The investigated embryos were categorized into two groups based on NPB clustering. We compared various parameters to identify differences between these groups. Continuous variables underwent analysis using the Student t-test, following confirmation of normal distribution and variance homogeneity. Categorical variables were evaluated using the chi-square test, and Fisher's exact test was applied when the expected embryo count fell below 5. All statistical analyses were conducted using SPSS version 12.0 (SPSS Inc.), with statistical significance established at p<0.05.
Results
1. Patient and clustering characteristics
Following fertilization, maternal and paternal chromosomes are enclosed within two distinct pronuclei located at the periphery of the zygote. These pronuclei then migrate toward the center of the zygote, where they meet and subsequently undergo PNBD and cell division. Prior to PNBD, NPBs move toward the interface between the two pronuclei (CL) (Figure 1A). However, in some instances, this movement does not occur, resulting in a failure of NPBs to cluster (NCL) (Figure 1B). Notably, at the time of PNBD, 51.5% of blastocysts had successfully clustered their NPBs at the interface of the two pronuclei (Table 1).
2. Differential time-morphokinetics: impact of nucleolar precursor body clustering on preimplantation embryo development
To investigate potential differences in the development of preimplantation embryos, a comparative analysis of time-morphokinetics was conducted between CL and NCL groups. Table 2 shows that TL imaging analysis identified a statistically insignificant correlation at several time points (t3, t4, t5, t6, t7, t8, cc2, and s2) between NPB clustering and early embryo development. However, significant differences were observed at the time to pronuclear fading (tPNf) and t2 (onset of the first cleavage), which are specifically associated with the initiation of the first cleavage (p<0.05). Notably, tPNf and t2 occurred earlier in the NCL group than in the CL group. Conversely, the NCL group showed a delayed onset of blastocyst formation and blastocyst expansion relative to the CL group, with the time to blastocyst recorded at 104.2 hours for CL and 107.4 hours for NCL, and time to expansion noted at 112.2 hours for CL and 115.3 hours for NCL (p<0.05).
3. Nucleolar precursor body clustering, unusual division patterns, and their impact on euploidy in preimplantation embryos
Abnormal division behaviors in preimplantation embryos were analyzed to study their correlation with NPB clustering (Table 3). No significant differences were observed between the CL and NCL groups in terms of uneven blastomere size (2.0% vs. 4.1%, p=0.43) and the rate of multinucleation (30.1% vs. 27.8%, p=0.73) at the 2-cell stage. Similarly, no significant differences were found in the rates of direct cleavage from 1- to 3-cell (0.0% vs. 3.1%, p=0.11) and from 2- to 5-cell (5.0% vs. 5.2%, p=0.92) between the CL and NCL groups. However, the frequency of fragmentation greater than 20% at the 8-cell stage showed a statistically significant difference between the CL and NCL groups (1.9% vs. 10.3%, p<0.05). Additionally, there was a significantly higher rate of euploid blastocysts in the CL group than in the NCL group (37.9% vs. 12.4%, p<0.05).
Discussion
The current study, conducted using TL data, has revealed a correlation between the kinetics of NPB clustering—an event that occurs during PN formation—and both morphokinetics and abnormal cleavage. Furthermore, extending this investigation, the association between NPB clustering and the occurrence of aneuploidy was demonstrated through the analysis of blastocysts from PGT-A cycles.
The observation of pronuclei migration and convergence before PNBD underscores the intricately coordinated nature of early embryonic events [18,19]. The convergence of nucleoli before PNBD plays a critical role in organizing the genetic material spatially [20-23]. Additionally, we demonstrated that the failure of NPBs to cluster at a specific time, as shown in Figure 1B, suggests a possible deviation from the typical developmental pathway. Our comprehensive analysis of TL videos indicated that approximately 51.5% of blastocysts in PGT-A cycles successfully clustered their NPBs at the interface of the two pronuclei at the time of PNBD. Interestingly, when comparing embryos with different NPB kinetics in the pronucleus, developmental differences were clearly evident between the CL and NCL groups. TL imaging analysis showed no significant correlation between NPB clustering and early embryo development, except at tPNf and t2. Notably, the NCL group exhibited earlier first cleavage but experienced delayed blastocyst formation and expansion compared to the CL group. These findings underscore the subtle interactions between NPB clustering and embryonic events, providing valuable insights for assisted reproductive technologies. Furthermore, the NPB clustering method may prove useful in clinical settings because the classification of CL and NCL is straightforward, clearly defined with TL images, and yields consistent assessments among embryologists.
The investigation into abnormal division behaviors during preimplantation embryo development offers intriguing insights. Notably, there were no differences between the CL and NCL groups in terms of uneven blastomere size or the rate of multinucleation at the 2-cell stage. However, the statistically significant difference in the frequency of fragmentation between the CL and NCL groups suggests that NPB clustering may affect the integrity of blastocysts and impact developmental outcomes.
It has been suggested that abnormal chromosomal segregation primarily occurs during the release of the first polar body, coinciding with cytoplasmic maturation [24,25]. In a recent study, Wang et al. [26] found no relationship between the aggregation of smooth endoplasmic reticulum in oocytes and chromosome aneuploidy in embryos resulting from IVF. It is noteworthy that a previous study by Cavazza et al. [17] identified a link between the failure of nuclei clustering and chromosome segregation errors. Such errors in chromosome segregation are known to lead to fragmentation, including the formation of micronuclei [27-32]. In conjunction with this study, it is suggested that the clustering kinetics of NPBs just before PNBD may be correlated. This finding highlights the significant impact that chromosome segregation errors, associated with nuclei clustering, can have on fragmentation and, consequently, on blastocyst developmental outcomes.
The limitations of this study stem from its retrospective nature and the relatively small sample size from a single clinic center. We cannot rule out the possibility of other unknown causes of aneuploidy that IVF may not effectively address. Despite these limitations, this comprehensive investigation, which utilizes TL technology and PGT-A, introduces a novel non-invasive predictive tool for use in ART. The results align with findings from other groups, highlighting the significant value of early embryonic events, including morphokinetics and abnormal division behaviors during cleavage. Additionally, the study underscores the importance of NPB kinetics in ART. Here, we expanded the analysis to include both NPB kinetics and morphokinetics, revealing a notable association with aneuploid occurrences, as evidenced by the analysis of blastocyst embryos from PGT-A programs. To further explore these findings in the context of clinical implications, additional well-designed experiments and more retrospective studies are necessary. Integrating the observed NPB clustering kinetics in the early post-fertilization stages with morphokinetics may provide crucial insights for selecting embryos with higher developmental potential and identifying blastocyst embryos with normal chromosomal content. This non-invasive tool could prove invaluable in optimizing assisted reproductive strategies.
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