Embryonic Arrest, Contributing Factors and Proposed Solutions: A Brief Review.

Couples trying to conceive may experience infertility after multiple attempts. They are considered infertile if the couple are unable to conceive, despite a year of continuous unprotected attempts. Assisted reproductive technologies (ARTs) are among the methods helping these couples to overcome their infertility problem. ARTs include intracytoplasmic sperm injection (ICSI), in vitro fertilization (IVF), and in vitro maturation (IVM), with ICSI and IVF being the most effective. These techniques involve retrieving appropriate oocytes by a specialist following ovarian stimulation. Then, these oocytes and sperms fertilize in vitro to form a zygote, which is transferred to the patient after go ing through several developmental stages ( 1 ). Embryonic development may be adversely affected by various disturbances, including altered gene expression, increased reactive oxygen species (ROS) levels ( 2 ), exposure to toxic substances ( 3 ), and suboptimal embryo culture conditions ( Fig .1 ) ( 4 ). Such disruptions may lead to embryonic arrest, characterized by a cessation of cell division for at least 24 hours, often resulting in failure of ART cycle success. Metabolically arrested embryo blastomeres may undergo complete degeneration or lysis due to decreased activity ( 5 ). Studies indicate that approximately 40-50% of embryos fail to develop to the blastocyst stage, frequently arresting at the two- to four-cell stage during early mitotic divisions, typically on days 3 or 4 of gestation ( 5 , 6 ). Interestingly, these arrested embryos often show no signs of apoptosis and have a lower incidence of aneuploidy compared to normally developing embryos ( 6 ). Arrested embryos. After embryo formation during IVF and ICSI processes, a percentage of the resulting zygotes develop normally and reach the blastocyst stage, whereas the remaining embryos fail to form a blastocyst and are arrested in the early stages of embryogenesis. ART; Assisted reproductive technologie, ICSI; Intracytoplasmic sperm injection, IVF; In vitro fertilization, and ROS; Reactive oxygen species. This overview aims to provide foundational insights into embryonic arrest, including its types, causes, and potential strategies to enhance ART success rates by addressing the developmental arrest phenomena. In the discussion section, we emphasize on understanding the arrest process and exploring several methods to overcome this issue to improve fertility treatments. Preimplantation embryo arrest (PREMBA) manifests in two forms: temporary and permanent. Temporarily arrested embryos do not exhibit any signs of developmental progress within the first 24 hours; however, they can resume development and reach the blastocyst stage after this period ( 7 , 8 ). Research suggests that advanced maternal age is associated with a higher incidence of temporary embryonic arrest ( 8 ), potentially due to compromised oocyte competence ( 9 ). Additionally, individuals undergoing ICSI tend to experience a greater rate of temporary arrest compared to those undergoing conventional IVF. Embryos in this category often develop into compact morulae or early blastocysts, with a lower proportion of blastocysts with expanded blastocoel cavities compared to normal embryos ( 8 ). In contrast, permanently arrested embryos fail to develop beyond early developmental stages, often showing no progression to the blastocyst stage even after 118 hours of culture ( 5 ). The classification of arrested embryos has historically posed significant challenges due to their complex and variable characteristics. To address this, Yang et al. ( 6 ) proposed a taxonomy dividing arrested embryos into three distinct categories: type I (arrest before the four-cell stage), type II (from the four-cell to early morula stage), and type III (from early to late morula stage). These embryos have been shown to retain both developmental potential and gene expression profile characteristic of the arrest stage ( 6 ). Type I arrested embryos, typically at the 2- to 3-cell stage, are unable to successfully execute the maternal-tozygotic transition (MZT) ( 6 ). Impaired MZT has also been documented in 2- 4 cell arrested embryos ( 10 ). During MZT, the inherited alleles are expressed through zygotic genome activation (ZGA), followed by the clearance of maternal transcripts. In these embryos, elevated expression levels of maternal genes such as PAN2, CNOT6L, and BTG4 persist, indicating defective maternal transcript degradation, an abnormality that normally diminishes during the 4-8-cell stages ( 6 , 11 ). Deregulated expression of epigenetic regulators and key genome activation genes, such as DUX4 and ZSCAN family members, is observed in arrested embryos ( 6 ). Dux4, a transcription factor, is activated just before major ZGA and plays a role in this process, with its target gene ZSCAN4 also being important for early embryonic development ( 12 ). In contrast, type II and III arrested embryos display metabolic deficiencies characterized by impaired glycolysis and oxidative phosphorylation, leading to a state of cell cycle quiescence. These embryos also exhibit reduced number of nucleosomes and ribosomes, further contributing to cell cycle arrest. Notably, genes that promote cell cycle progression, such as CCNA2 , are downregulated, whereas inhibitors like CDKN1A (P21), along with P53 activity and FOX mRNA levels, are upregulated ( 6 ). Given our limited understanding of the molecular mechanisms underlying early developmental arrest, further research is essential in this area. Comparing RNA sequences on day 3 post-fertilization between arrested (≤5 cells) and normal (6-8 cells) embryos revealed that approximately 66% of the genes with altered expression were downregulated in the arrested embryos. Notably, significant modifications in processes such as epigenetics, ubiquitination, cell cycle regulation, signaling pathways, metabolism, cell proliferation, adhesion, differentiation, fate determination, energy homeostasis, and transcription were observed in these embryos compared to their normal counterparts. Additionally, most hub genes involved in early embryonic development, were downregulated in the arrested embryos. Disruption of these genes leads to cell cycle arrest and ultimately halts preimplantation embryonic development ( 13 ). Disruption of the hub genes, along with reduced cell cycle activity and energy metabolic pathways, has also been documented in other species experiencing embryonic arrest at the 2- to 4-cell stage ( 10 ). Permanent cell cycle arrest results in increased cell inflammation, decreased cell proliferation, and subsequently, cell aging ( Fig .1 ) ( 14 ). Disruption of the cell cycle is a primary mechanism underlying embryonic arrest ( 10 ), as discussed in Yang et al. ( 6 ). These embryos have been shown to face downregulation of CCNA2 (a promoter of cell cycle progression) and upregulation of CDKN1A (a cell cycle inhibitor). These observations emphasize the critical importance of tightly regulated cell cycle mechanisms during embryogenesis, as proper regulation allows for effective responses to DNA damage and prevents developmental disruptions ( 15 ). Fertilization failure or impairment, such as mutations in WEE2, represents another leading cause of arrest ( 16 ). Different ARTs are recommended depending on the underlying cause be it genetic, epigenetic, or chromosomal abnormalities ( 17 ). Additionally, impaired DNA repair capacity may contribute, given that early embryos use maternal resources for DNA repair, with human oocytes showing high levels of gene expression repair. Severe paternal DNA damage in mouse zygotes triggers a non-apoptotic response, delaying replication and causing developmental arrest. This underscores the importance of DNA damage response in early embryonic survival ( 18 ). Methylation patterns of critical genes such as H19 , NANOG , and OCT4 ( 19 ), along with metabolic markers, like Caspase ( 20 ), are also influential factors in modulating developmental arrest. Signaling pathways are critical for proper development of an embryo. Even the slightest alteration in these pathways can disrupt developmental processes and result in embryo arrest ( 21 ). In this section, we will evaluate several signaling pathways that have been demonstrated in the literature to significantly influence both embryonic development and the potential for developmental arrest. Transcriptome and small RNA sequencing conducted on human embryos in a state of arrest indicated that disturbances in the PI3K-AKT, JAK-STAT, MAPK signaling pathways play a significant role in the arrest process ( 22 ). In the TGF-β signaling pathway, the regulation of the ID3 and BMP4 genes differs between normal and arrested embryos ( 23 ). Additionally, the accumulation of GPC3 and BRINP3 , both of which respond to TGF-β , negatively im pacts the cell cycle in arrested human embryos by inhibiting the transition from G1 to S phase ( Fig 2 ) ( 24 ). Signaling pathways extend beyond embryonic development, and their disruption may result in diseases that contribute to infertility. One such pathway is the Wnt signaling pathway. Research has demonstrated that this pathway is dysregulated in endometriosis, which can lead to female infertility in acute cases ( 25 ). Furthermore, heightened activity of this pathway has been linked to the presence of uterine fibroids, wherein elevated levels of β-Catenin are observed ( 26 ). Additionally, a notable mutation identified in infertile women pertains to the MOS gene, which functions upstream of the ERK pathway. Disruption of this gene is associated with a negative regulatory effect on ERK signaling ( 27 ). The role of signaling pathways in PREMBA is critical and encompasses a wide array of pathways that warrant further exploration, particularly in the context of IVF arrest. The influence of chromosomal abnormalities, such as mosaicism, haploidy, aneuploidy, and polyploidy, on the arrest of embryonic development during the IVF process remains a topic of considerable debate. Several studies have shown that chromosomal abnormalities are among the most prominent problems in preimplantation arrested embryos, which are likely to prevent further embyonic development ( 19 ). Research indicates that the prevalence of chromosomal abnormalities in embryos derived from mature oocytes in vivo is lower than that observed in vitro ( 28 ). Additionally, it has been noted that over 70% of embryos from older maternal age reaching the blastocyst stage are aneuploid ( 29 ). This situation highlights the influence of ART on the incidence of chromosomal abnormalities. It is essential to investigate whether chromosomal abnormalities may be recognized as a primary factor contributing to the embryonic developmental arrest (EDA). Paradoxically, while some studies suggest that aneuploidy does not significantly influence the occurrence of EDA ( 6 ), others identify it as a principal factor contributing to such arrests ( 30 ). Notably, more than 60% of embryos that undergo arrest following IVF, are found to possess chromosomal abnormalities, with a higher incidence of polyploidy compared to haploidy ( 31 ). Additionally, older maternal age is associated with an increased prevalence of both aneuploidy and arrested embryos ( 29 ). It is noteworthy that approximately 90% of the arrested embryos exhibit aneuploidy, which significantly impairs subsequent cleavage and leads to EDA ( 30 ). In general, chromosomal abnormalities may if fact be considered as significant contributing factors in the context of EDA. Mitochondria, which are inherited maternally, are crucial organelles within cells and significantly influence reproductive outcomes and embryonic development. While mitochondrial function is essential throughout various stages of embryo development, their primary and indispensable function is predominantly associated with oocyte metabolism ( 32 ). Disruption of mitochondrial integrity may lead to increased reactive oxygen species (ROS) production and a diminished mitochondrial membrane potential. Furthermore, such disturbances may induce apoptosis, adversely affecting connexins and cadherins, thereby impairing communication between oocytes and cumulus cells. This disruption ultimately contributes to ovarian depletion and may result in infertility ( 33 ). Numerous studies suggest that ART may have an impact on the mitochondrial status in the cell. The arrest may be attributed to the regulation of mitochondrial activity ( 19 ), as IVF embryos with reduced mitochondrial membrane potential are usually arrested after the 2-cell stage ( 34 ). Such disturbances may alter the distribution patterns of mitochondria ( 35 ) and increase mitochondrial permeability, thereby contributing to EDA ( 36 ). Nuclear transfer represents a viable solution for addressing such challenges and has demonstrated promising outcomes in the treatment of infertility ( 37 ). An essential aspect of developmental biology is the precise regulation and expression of various maternal, paternal, and embryonic genes. Disruptions in these genes may lead to diverse effects on embryonic maturation, potentially arresting development at any stage from a single-cell zygote to a morula or blastocyst formation ( Fig .3 ). Maternal gene disruptions, such as mutations in PADI6, TLE6, NLRP2, NLRP5, NLRP7, OOEP, KHDC3L, ZBED3, and NLRP4F ( 5 ) along with follicular factors like PTGS2 ( 38 ), IGFII, IGFPB and PAPP-A ( 39 ), as well as female-related conditions such as endometriosis ( 19 ) and diminished ovarian reserve (DOR) ( 40 ), also contribute to EDA. Preimplantation embryos may experience arrest at various stages of development, either at early stages (left panel) or at later stages (right panel), each exhibiting distinct characteristics. Genes involved in embryonic arrest. Any disruption in these genes can prevent the proper and on-time embryonic development, leading to temporary or permanent arrest. On the paternal side, genetic factors, including mutations in the PCSK4 gene may cause infertility due to sperm abnormalities ( 41 ). Additionally, factors such as chromatin condensation abnormalities ( 19 ), increased rates of apoptotic DNA fragmentation ( 42 ), and male-specific conditions, like azoospermia ( 19 ), may lead to developmental arrest. Furthermore, disruptions in the balance of histones and protamines during spermatogenesis, particularly defects in various histone variants, have been shown to be associated with reduced fertility ( 43 , 44 ), suggesting their potential roles in embryonic development. Genetic alterations may also impact the embryonic genome, potentially leading to developmental arrest ( 5 ). The SCMC is a crucial multi-protein complex, composed of PADI6, TLE6, KHDC3L, OOEP, and NLRP2 and NLRP5. Among these, PADI6 is implicated in EGA and genomic imprinting ( 5 ), therefore, its disruption prevents embryo formation beyond the 2- to 7-cell stages ( 45 ). Disruption of TLE6, which plays a significant role in cleavage and meiosis ( 5 ), may result in developmental arrest at the 1- to 9-cell stages ( 46 ). KHDC3L, another member of this complex, plays an effective role in spindle assembly ( 47 ), DNA repair ( 48 ), and embryonic development, and its disruption inhibits development at the morula stage ( 45 ). In addition, the disruption of OOEP, which is involved in the regulation of cytoplasmic network formation, symmetric division, and DNA methylation ( 5 ), interrupts development at the 2- to 6-cell stages ( 49 ). Moreover, NLRP2 is involved in the cellular processes of apoptosis, cell survival, and early embryogenesis ( 5 ), and its disruption affects the 2- to 7-cell stages of development ( 50 ). Finally, the disruption of NLRP5, which is involved in early embryogenesis and mitochondrial function ( 51 ), affects the 1- to 8-cell stages of development ( 49 ). The spindle structure also has a significant impact onthe cell condition and ultimately the embryo. The protein TUBB8 contributes to microtubule formation and spindle assembly, and plays a significant role at various stages of preimplantation development. Disruption of this protein has been shown to cause EDA ( 52 ). Besides, the protein associated with PATL2 promotes translational control ( 5 ) and is mainly active at the 2-cell stage ( 53 ). The disruption of MOS, which is involved in mRNA clearance during MZT, mitochondrial activity ( 54 ), MII phase regulation ( 55 ), and ERK signaling pathway ( 55 ), impacts the 2- to 8-cell stages of development ( 56 ). Cell cycle regulation is also critical for embryonic development. For instance, CDC20 plays an important role in the suppression of apoptosis ( 5 ) as well as the regulation of cyclin function ( 57 ). It also functions as a co-activator for the APC/C ( 58 ) and is mainly active at 4- to 5-cell stages of development ( 59 ). TRIP13, on the other hand, is involved in the regulation of the mitotic checkpoint and the creation of DNA double-strand breaks (DSBs) during meiotic recombination ( 5 ). It is primarily active at 1- to 8-cell stages, and its disruption may affect any of these stages ( 60 ). In addition, REC114, which affects 2- to 6-cell stages ( 61 ), is involved in the formation of DSBs during DNA recombination ( 5 ). Analyzing gene expression changes in embryos experiencing developmental arrest provides valuable insights into the underlying mechanisms and processes involved, aiding in the classification of causes and the development of targeted interventions. The conditions within the embryology laboratory and the composition of the culture media used for embryo cultivation [ in vitro maturation (IVM) or IVF media] are critical factors influencing optimal embryonic development. While culture media aim to meet the nutritional and environmental requirements of the gametes and the embryos, their suitability may vary, as inappropriate media may impair embryonic growth ( 62 ). For instance, inadequate pyruvate concentrations in the culture medium have been associated with developmental arrest ( 63 ). Conversely, the addition of granulocyte macrophage-colony stimulating factor (GM-CSF) to the culture medium has been shown to improve reproductive outcomes in patients experiencing IVF failure and embryonic arrest ( 64 ). ROS are continually generated during cellular metabolism within the culture media, and excessive ROS levels can induce cellular aging through processes such as differentiation and apoptosis ( 65 ). The concentration of ROS in follicular and embryonic cultures directly influences developmental potential ( 66 ). ROS can be produced due to suboptimal oxygen tension, contamination in laboratory settings, the materials used in ART such as culture plates and plastics, temperature fluctuations, supplementation in culture media (e.g., mineral oil), IVF procedures, and cryopreservation processes ( 67 ). Additionally, sperm-derived ROS, particularly mitochondrial ROS and hydrogen peroxide, significantly impact embryonic development; as elevated levels are associated with impaired blastocyst formation. Therefore, monitoring sperm ROS levels may enhance IVF success rates. Importantly, the ROS levels in high-quality embryos are markedly lower than those in embryos that exhibit developmental arrest, emphasizing the importance of ROS regulation in embryogenesis ( 68 ). In summary, optimizing the composition of the culture media to meet the embryo’s nutritional needs, while minimizing the production of harmful reactive species, is essential for improving embryonic outcomes. Proper regulation of these components supports the development and increases the success rates of ARTs. ART laboratories may, albeit rarely, encounter procedural errors or systemic challenges that can impact embryonic development. These issues can be mitigated or prevented through proactive planning and the implementation of a robust management system. Ensuring adequate quality control within an embryology laboratory is vital to maintaining a stable and optimal environment conducive to embryogenesis. This involves regular assessment of equipment performance and meticulous monitoring of the environmental parameters such as temperature, pH, CO 2 levels, air quality ( 69 ). Furthermore, maintaining appropriate culture conditions, utilizing high-quality plasticware, and preventing chemical toxicity, are essential components. Minimizing unnecessary handling of the embryos also contributes to reducing potential developmental disruptions. Strict adherence to quality standards minimizes the risk of developmental abnormalities and enhances overall success rates in assisted reproductive procedures ( 70 ). Moreover, protocols aligned with guidelines, such as those established by ASPIRE, help improve laboratory workflows and elevate the quality of management systems within the ART settings ( 71 ). Ensuring optimal conditions during embryonic development is also paramount to achieving successful results ( 72 ). Several critical steps within the embryology and IVF laboratory may, if improperly managed, lead to embryo arrest ( 72 ). These include ovarian stimulation protocols, oocyte retrieval techniques, and the proficiency of the surgeon factors that influence oocyte quality ( 73 ), such that high-quality oocytes are less prone to arrest and developmental failure. Accurate labeling is a fundamental, yet often overlooked aspect; errors in labeling at any stage from fertilization to cryopreservation are significant sources of potential mistakes ( 74 ). Such errors may result in oocytes with limited fertilization potential being misclassified as high-quality, thereby adversely affecting embryo development. Oocyte cryopreservation is a common method for fertility preservation and is frequently utilized in ART laboratories. However, due to the high water content in oocytes, which elevates the risk of ice formation and cellular damage ( 75 ), this method presents greater technical challenges compared to embryo cryopreservation. Over the recent decades, embryo cryopreservation techniques have become safer and more effective, with rapid freezing and vitrification methods replacing traditional approaches. These advancements have significantly improved survival rates and clinical outcomes ( 76 ). Successful vitrification depends on precise control of such parameters as the concentration of cryoprotective agents and the rates of cooling and warming. While this technology effectively prevents ice crystal formation and the consequent cellular injury, its success remains influenced by the initial quality of the cells. Currently, optimizing preservative formulations and their application concentrations plays a critical role in enhancing vitrification outcomes ( 77 ). The complex nature of the freeze/ thaw process requires meticulous adherence to the available protocols, high-quality materials, and optimal environmental conditions within the laboratory, to ensure cell integrity and maximize success rates. Recent studies highlight potential adverse effects of nanoparticles on reproductive health and embryonic development. Despite their numerous benefits, nanoparticles, such as zinc oxide (ZnO NPs), have been shown to impair mammalian oocyte maturation and pre-implantation embryo development by inducing oxidative stress, mitochondrial and endoplasmic reticulum dysfunction, and cytoskeletal disruption ( 78 ). Similarly, chitosan nanoparticles (CSNPs) negatively affect blastocyst formation and viability, resulting in reduced cell numbers and increased apoptosis. These findings underscore the need for further evaluation of human exposure risks ( 79 ). In summary, maintaining strict adherence to laboratory protocols, implementing quality management systems, and considering individual patient conditions are crucial for the success rate of the ART procedures ( 72 , 74 ). Table 1 lists some reasons for developmental arrest, along with potential solutions to overcome this phenomenon. In general, in laboratory conditions, the integrity of rules and guidelines, as well as their correct implementation and the patient’s condition, are crucial for the success of a treatment cycle. Reasons of PREMBA PREMBA; Preimplantation embryo arrest, TCA; Tricarboxylic acid cycle, mtDNA, ROS; Reactive oxygen species, and iSCNT; Interspecies somatic cell nuclear transfer. Different variants of the genes mentioned above, may experience mutations or defects. Therefore, the strategies to counteract their effects and ensuing disorders have been investigated. Assessing oocyte morphology prior to fertilization, although potentially useful for predicting treatment outcomes, is not yet widely practiced due to its methodological complexity and the need for advanced technologies, such as artificial intelligence. Morphological abnormalities may influence the embryo’s ability to grow and implant, but their definitive effects require further confirmation ( 88 ). More over, oocytes increasing the probability of embryonic arrest, can be identified and excluded from ART cycles at earlier stages (i.e. before fertilization and arrest induction), to reduce the chance of formation of arrested embryos. Oocyte quality, not only in terms of cytoplasmic contents and biochemical capabilities (such as DNA repair and cell cycle control), but also in terms of mechanical properties, plays a pivotal role in determining the growth potential and viability of the embryo. Yanez et al. ( 81 ) evaluated the embryo developmental potential using a minimally invasive mechanical method. Mechanical properties, which may be obtained by measuring the depth of aspiration and fitting the data to a mathematical model, are the basis for separation. This is because certain values of these parameters, e.g. embryo stiffness, elasticity, and viscosity, have been experimentally correlated with embryo viability, as defined by reaching the blastocyst stage or arresting growth. Thus, researchers can measure these properties and divide embryos into “predicted viable” and “predicted nonviable” categories and then transfer the viable and non-viable oocytes to mice to examin their differences. The results observed by Yanez et al. ( 81 ) indicated a significant decrease in cell cycle checkpoint control and chromosome segregation in the non-viable embryos. Besides, the viable group exhibited a higher survival rate than both the non-viable and the control groups. The comparison results indicated that the cortical granule release rate (indicated by cortical granule staining) in the low-quality oocytes was impaired compared to the higher-quality oocytes. Additionally, the quality and morphology of sperm also play a significant role in ART outcomes, particularly in ICSI; sperm with a normal appearance tend to have higher fertilization and pregnancy rates, while decreased semen quality impacts parameters, such as fertilization and cell division. Nevertheless, these changes do not directly affect the final pregnancy outcome. Therefore, precise evaluation of both factors could help improve infertility treatment strategies, but ongoing research and designing more accurate methods are still needed ( 82 , 83 ). One of the recent strategies employed in human studies involves enhancing the culture environment to overcome problems related to arrested embryo. A study on arrested human embryos explored the effects of resveratrol, rapamycin, vitamin C, and PD032 on arrest rate. Among these medications, resveratrol could reduce the arrest rate by 45% and reach the blastocyst stage in 7% of the embryos, expressing genes in this stage, while genes related to cell cycle activity still showed low expression levels ( 6 ). A recent study conducted by Karami et al. demonstrated that the addition of the small molecule CHIR99021 and insulin transferrin selenium (ITS) to type II arrested embryos significantly enhances developmental rates. Specifically, the study observed that the proportions of embryos achieving the blastocyst stage in those treated with CHIR99021 and ITS were 12.5 and 17.5%, respectively. Furthermore, ITS demonstrated the capacity to induce a normal state of pluripotency, as well as gene expression associated with the cell cycle ( 7 ). On the other hand, activating arrested oocytes with calcium ionophore may increase clinical pregnancy and live birth rates in humans ( 85 , 89 ), but has no significant effects on miscarriage rates and sex ratio ( 85 ). Liu et al. ( 86 ) demonstrated that microinjection of recombinant BCL-XL into mouse oocytes using an automated robotic system prevented cell cycle arrest in embryos cultured under unfavorable conditions, by inhibiting ROS accumulation and mitochondrial repair. High oxygen levels are likely to induce permanent arrest in preimplantation bovine embryos by increasing P66shc gene activity, but not P53. A group of these embryos cultured in a 20% oxygen medium compared with a group cultured in a 5% oxygen medium experienced a 10-fold increase in oxidative stress, which ultimately resulted in a twofold increase in the percentage of 2- to 4-cell arrested embryos ( 87 ). They then used RNA interference to significantly reduce the rate of 2- to 4-cell embryo arrest by reducing P66shc mRNA levels ( 90 ). In light of the aforementioned considerations, it is essential to determine the specific cause of any developmental arrest in embryos in order to implement an effective solution. Preliminary evidence suggests that enhancing the culture conditions for arrested human embryos may yield promising results; however, further comprehensive studies are required to validate these findings. Research involving human arrested embryos faces several significant technical limitations. Firstly, the availability of these embryos for research purposes is inherently restricted, which can impede the progress of large-scale and comprehensive studies ( 7 ). Secondly, the quality of arrested embryos is often compromised, with potential cellular and genetic damage, thereby affecting the reliability and generalizability of the research findings. Furthermore, the arrested embryos donot fully model the intricate complexities of normal embryonic development, which limits our understanding of biological processes and disease mechanisms. Lastly, the in vitro culture and maintenance of human embryos, particularly in early developmental stages, present considerable challenges due to the incomplete understanding of optimal growth conditions, potentially leading to unintended changes in the embryos. Successful reproduction hinges on the precise regulation of fertilization and early embryonic development, which is influenced by a complex interplay of internal and external factors. Disruptions in these factors may lead to embryonic arrest, posing a significant obstacle in both natural conception and ARTs, such as IVF and ICSI. Addressing this issue requires a deeper understanding of the molecular mechanisms involved, leveraging advances in embryo culture conditions, intracellular signaling pathways, and gene expression modulation. Identifying and targeting genetic variations and dysfunctions help mitigate developmental arrest, although current assessment methods, such as evaluating oocyte and sperm morphology, face limitations due to their complexity and technological demands. The decision to allow arrested embryos to resume development raises important ethical and scientific questions: should embryos always be forced to develop, even if they lack the competence to produce a viable human? Could such interventions lead to unforeseen problems? Therefore, ongoing research aims to balance the desire to improve embryo viability with ethical considerations, seeking to develop more accurate evaluation techniques and clinical strategies. Ultimately, a comprehensive approach that simultaneously investigates causes, signaling pathways, and effective treatments, is essential for advancing ARTs, improving success rates, and addressing infertility challenges.