The large-scale production of mammalian cells is pivotal for various applications; however, current bioreactor technologies encounter significant technical and economic challenges. Scaling up cell cultures remains problematic due to excessive cell aggregation, shear stress-induced cell death, batch-to-batch inconsistencies, and limited scalability. We propose that engineering a cell-friendly microenvironment can enhance culture efficiency. Previously, we developed alginate hydrogel microtubes (AlgTubes) that significantly improved cell density and growth rates. Nevertheless, AlgTubes lack adhesion sites essential for anchorage-dependent cells and frequently break, causing cell leakage and production inconsistencies. To address these limitations, this study reinforced AlgTubes with collagen nanofibers, creating collagen-alginate hybrid hydrogel microtubes (ColAlgTubes). Utilizing a novel micro-extruder, we efficiently produced cell-loaded ColAlgTubes. Collagen formed a dense nanofiber network interwoven with the alginate mesh, enhancing the hydrogel's mechanical properties while providing cell adhesion sites. ColAlgTubes protected cells from hydrodynamic stress and maintained cell mass within a 400 μm diameter, ensuring efficient nutrient exchange and waste removal. This optimized microenvironment resulted in high cell viability, rapid proliferation, and exceptional yields of 5×10 8 cells/mL - 200 times higher than conventional culture methods. With their scalability, cost-effectiveness, and efficiency, ColAlgTubes offers a transformative solution for large-scale cell production with broad applications in biotechnology, regenerative medicine, and therapeutic manufacturing.

Although essential to confirm the clinical utility of direct cleavage embryos, the timing of rapid cleavage (RpiC) has not been specifically defined. This study aimed to explore the differences in embryonic and clinical outcomes based on varying timing parameters of direct cleavage during first cytokinesis, using time to reach the three-cell stage (t3) minus time to reach the two-cell stage (t2). We analyzed 19,796 fertilized embryos (in 6,907 patients) from a single center between September 2019 and December 2020. The embryos were cultured using EmbryoScope, and t2 and t3 were recorded. Trichotomous mitosis (TM) was defined as t3 - t2 = 0 h, and RpiC events were divided into four groups, as follows: 0 h < t3 - t2 < 1 h (RpiC-1), 1 h ≤ t3 - t2 < 3 h (RpiC-3), 3 h ≤ t3 - t2 < 5 h (RpiC-5), and 5 h ≤ t3 - t2 < 7 h (RpiC-7). Additionally, 7 h ≤ t3 - t2 < 14 h was defined as normal cleavage. After single-cleavage embryo transfer, the live-birth, TM, and RpiC-1 rates were significantly lower than those in other groups. Similarly, when blastocysts were utilized, the TM and RpiC-1 rates were significantly lower than those in other groups. This study suggests that embryos with TM or RpiC-1 (t3 - t2 < 1 h) should be cultured to the blastocyst stage to prevent unnecessary embryo transfers, although outcomes may vary in different scenarios, i.e., by institution and patient.

Preimplantation aneuploidy in humans is one of the primary causes of implantation failure and embryo miscarriage. This study was conducted to gain insight into gene expression changes that may result from aneuploidy in blastocysts through RNA-Seq analysis.

The surplus embryos of preimplantation genetic testing for aneuploidy (PGT-A) candidate couples with normal karyotype and maternal age < 38 were collected following identical ovarian stimulation protocol. The embryos were selected based on trophectoderm biopsy and array comparative genomic hybridization in three groups: normal group, small chromosomes aneuploidy group (SCA), including single aneuploidy for small chromosomes 16, 20, 21, 22, and other chromosomes aneuploidy group (OCA), including single aneuploidy for other chromosomes.

Principal component analysis revealed overall differentiation of transcriptome of the groups, confirming embryo classification. The Gene Ontology indicated that transcription, ubiquitination, autophagy, and DNA repair pathways were upregulated in aneuploid embryos. The overexpression of five genes, UBE2E2 and VPS4A, BUB1B, CDCA8, and COX14 was confirmed by quantitative real-time PCR. Additionally, overexpression was observed in translation and protein synthesis pathways in aneuploid embryos. Mitochondrial pathway upregulation was notable in both SCA and OCA groups, while the apoptosis pathway was overexpressed only in the OCA group. Only cellular lipid synthesis pathway differed between SCA and OCA, the two aneuploid groups.

This study highlights the impact of aneuploidy on the gene expression in blastocysts independent of aneuploidy type and paves the way for understanding the molecular mechanisms underlying the generation of aneuploidy.

The primary objective of this study was to develop predictive models for the likelihood of live births following In Vitro Fertilisation (IVF) treatment, based on a retrospective analysis of time-lapse data from Day 2 embryo transfers at Klinikk Hausken, Norway. This analysis encompassed 1,506 IVF treatment cycles, which included 865 single and 641 double embryo transfer cycles, totalling 2,147 embryos transferred. The model covariates included nucleation error, timing of two-cell stage (t2) and duration between t2 and the three-cell stage (t3). The predictive ability was assessed using Area Under Curve (AUC). Generalised Additive Mixed Models (GAMM) were utilised to address clustering effects from Single Embryo Transfers (SET) and Double Embryo Transfers (DETs), as well as the non-linear effects of female age and t2 timings. A stratification of age and model scores demonstrated the impact of incorporating age into the model. The" Base Model, not incorporating age, achieved an AUC of 0.641, while the "Age Model", using maternal age, significantly enhanced AUC to 0.745, as estimated through bootstrap analysis. However, when the Age Model was subjected to average ages across three respective age intervals, the AUC values were comparable to the Base Model, rather than the original Age Model scores. Adjusting the Intracytoplasmic Sperm Injection (ICSI) timing by ± 2 hours, purely as a theoretical exercise, has minimal impacts on model predictions. This highlights the value of including t2 despite fertilisation timing variations between ICSI and IVF. The Age Model did not show superiority in predicting live birth within single treatment cohorts. However, given its distinct AUC values for broader age ranges, the Age Model can serve as a counselling tool on live-birth probabilities. With further validation, we suggest only using the Age Model for general counselling, while the Base Model is preferable for the embryo selection decision support.

Discourses on human embryo experimentation often refer to monozygotic twinning and individuation. A criterion to establish regulations that guide human embryo research proposes that individuation is achieved once the embryo ceases to have the potential for dividing into two or more viable entities at about 15 days of gestational age. This standard is based on an updated version of a developmental model initially proposed by George Corner. A fundamental problem with this approach is the model's lack of sufficient evidence to explain adequately human embryo twinning and, consequently, to serve as a basis to establish appropriate ethical guidelines for embryo experimentation. In addition, subsequent formulations of Corner's model added an extension of blastomere totipotency to different moments of gestation, without a proper scientific basis. The model is also challenged by monozygotic twinnings that result in placental and amniotic arrangements incompatible with Corner's framework. Investigations into the physiology of fertilisation and of the zygote suggest that individuation may occur at a very early stage. An alternative description of monozygotic twinning may explain better sesquizygotic twinning events and serve to re-evaluate the individuation criterion. The study aims to investigate deficiencies in the embryology of this model and assess their ethical implications.

Recent advances in embryology have shown that the sister blastomeres of two-cell mouse and human embryos differ reciprocally in potency. An open question is whether the blastomeres became different as opposed to originating as different. Here we wanted to test two relevant but conflicting models: one proposing that each blastomere contains both animal and vegetal materials in balanced proportions because the plane of first cleavage runs close to the animal-vegetal axis of the fertilized oocyte (meridional cleavage); and the other model proposing that each blastomere contains variable proportions of animal and vegetal materials because the plane of the first cleavage can vary - up to an equatorial orientation - depending on the topology of fertilization. Therefore, we imposed the fertilization site in three distinct regions of mouse oocytes (animal pole, vegetal pole, equator) via ICSI. After the first zygotic cleavage, the sister blastomeres were dissociated and subjected to single-cell transcriptome analysis, keeping track of the original pair associations. Non-supervised hierarchical clustering revealed that the frequency of correct pair matches varied with the fertilization site (vegetal pole > animal pole > equator), thereby, challenging the first model of balanced partitioning. However, the inter-blastomere differences had similar signatures of gene ontology across the three groups, thereby, also challenging the competing model of variable partitioning. These conflicting observations could be reconciled if animal and vegetal materials were partitioned at the first cleavage: an event considered improbable and possibly deleterious in mammals. We tested this occurrence by keeping the fertilized oocytes immobilized from the time of ICSI until the first cleavage. Image analysis revealed that cleavage took place preferentially along the short (i.e. equatorial) diameter of the oocyte, thereby partitioning the animal and vegetal materials into the two-cell blastomeres. Our results point to a simple mechanism by which the two sister blastomeres start out as different, rather than becoming different.

Intracytoplasmic sperm injection (ICSI) is the current clinical practice for the in vitro production of equine embryos. The use of conventional fertilization methods such as in vitro fertilization (IVF), has historically been associated with poor success in horses. However, recent improvements have led to better outcomes with IVF, though only when using fresh semen, which limits its use in clinical practice. IVF remains in its infancy in equine reproduction, and several unknowns remain about the technique. One significant gap in knowledge concerns the morphokinetics of IVF embryos and how they differ from their ICSI counterparts. To address this, we performed IVF using frozen-thawed sperm from five different stallions following sperm selection and a prolonged capacitation period of 10 h, on a total of 109 oocytes. We then analyzed the cleavage rate (cleaved/initial oocytes), blastocyst rate (blastocyst/initial zygotes), and blastocyst development (blastocyst/cleaved zygotes) of the IVF cycles, and compared them with those of the clinical ICSI cycles during the same period. We also evaluated time-lapse images of the developed embryos to assess developmental time points such as time to morula compaction and blastocyst expansion, as well as morula and blastocyst sizes. Overall, developmental rates were not different between IVF and ICSI cycles (blastocyst rate 41.1 % IVF and 41.8 % ICSI, p > 0.05). However, development proceeded faster in IVF cycles (blastocyst expansion IVF 155.5 ± 18.5 h; ICSI 167.2 ± 19.6 h; p < 0.05) and IVF embryos were also larger (blastocyst area IVF 22608 ± 2857 μm2; ICSI 20806 ± 1505 μm2; p < 0.05). The faster development and larger size might suggest a more advanced developmental stage. The implications of these findings need to be further evaluated to assess their association with pregnancy potential. The successful developmental rates achieved in IVF cycles demonstrate the potential of this technique for clinical application, although the amount of frozen-thawed semen required is significantly higher in IVF than in ICSI, which is an important consideration for mare and stallion owners. Nonetheless, the use of frozen-thawed semen in equine IVF, coupled with comparable blastocyst rate, presents promising potential for broader clinical adoption of the IVF technique.

The production of mammalian cells in large quantities is essential for various applications. However, scaling up cell culture using existing bioreactors poses significant technical challenges and high costs. To address this, we previously developed an innovative 3D culture system, known as the AlgTube cell culture system, for high-density cell cultivation. This system involves processing cells into microscale alginate hydrogel tubes, which are suspended in the culture medium within a vessel. These hydrogel tubes shield cells from hydrodynamic stress and maintain the cell mass below 400 µm in diameter, facilitating efficient mass transport and creating a favorable microenvironment for cell growth. Under optimized conditions, AlgTubes supported long-term culture with high cell viability, rapid expansion (1000-fold increase over 9 days per passage), and high yield (5×10⁸ cells/mL), offering significant advantages over conventional methods. Despite these benefits, AlgTubes have critical drawbacks. They are mechanically fragile, with frequent breakage during culture leading to cell leakage and production failures. Additionally, many cell types exhibit poor growth due to the inability to adhere to the alginate surface, making alginate hydrogel microtubes unsuitable for industrial-scale cell production. To overcome these challenges, we developed a novel collagen hydrogel tube-based microbioreactor system in this work. This system provides enhanced robustness and adhesion, enabling scalable, cost-effective, and efficient cell production for a wide range of applications.

A time-lapse live embryo monitoring system provides a powerful approach to recording dynamic developmental events of cultured embryos in detail. By obtaining continuous short-interval images, blastocyst formation can be predicted and embryos can be selected. The objective of this study was to investigate the morphokinetic parameters of fishing cat-domestic cat interspecies somatic cell nuclear transfer (iSCNT) embryos from one-cell to blastocyst stages, and in particular, the cleavage patterns of the first division in iSCNT and IVF embryos, as these play a central role in euploidy. Domestic cat in vitro fertilization (IVF) embryos were set up as controls. The results show that morula and blastocyst development rates were significantly lower in the iSCNT embryos compared to their IVF counterparts. All earlier time points of embryonic development before the onset of blastulation in the iSCNT embryos were significantly delayed when compared with their IVF counterparts. In iSCNT, normal embryos (defined as those that developed to the blastocyst stage) took a longer time to reach the morula stage, and these morulas were more likely to undergo compaction, compared to their arrested embryo counterparts. Direct cleavage in the first division is a morphological aberration, and was seen with greater prevalence in iSCNT embryos than control IVF embryos; these aberrant embryos displayed a significantly lower blastocyst development rate than embryos that had undergone normal cleavage. In conclusion, the morphokinetic parameters of fishing cat-domestic cat iSCNT embryos at early stages could be used to predict their potential for development to the blastocyst stage. A time-lapse imaging system is potentially a powerful tool for selecting early embryos with developmental potential for transfer, and hence, for improving feline iSCNT efficiency.

Female infertility is a significant healthcare burden that is frequently encountered among couples globally. While environmental factors, comorbidities, and lifestyle determine reproductive health, certain genetic variants in key reproductive genes can potentially cause unsuccessful pregnancies. Such crucial proteins have been identified within the subcortical maternal complex (SCMC) and play an integral role in the early stages of embryogenesis before embryo implantation. SCMC proteins are associated with crucial pathways during embryogenesis, causing changes that are necessary for the transition of an oocyte to an embryo. These vital processes include the formation of cytoplasmic spindles and lattices, accurate positioning of meiotic spindles, regulatory roles in various gene translations, organelle redistribution, and zygotic genome reprogramming. While these genes are well studied in animal models, often mice, translation to clinical studies is comparatively less. The present study elucidates the transition in genetic studies from animal to human models of SCMC proteins. The present literature review shows that the expression of various SCMC proteins impairs embryo development at different stages. The clinical translation of SCMC occurs via various pathways. Therefore, females experiencing multiple unsuccessful pregnancies after natural or assisted conception techniques are candidates for underlying SCMC mutations. Although the phenotype of affected individuals has been identified, the molecular mechanisms that lead to impaired pathways still require investigation. Therefore, the present study paves the way for future research leading to the early diagnosis of lethal variants and possible subsequent management.

Recent evidence showed that the phase between pronuclear fading and the first cleavage is a perilous bridge connecting the zygote and the embryo. Indeed, delay in the short interval between pronuclear breakdown (PNBD) and the first cytokinesis may result in chromosome segregation errors. We tested the hypothesis that delays in this final phase of fertilization are associated with a detrimental impact on embryo development.

This is a retrospective study of 1315 zygotes cultured using time lapse technologies generated in 205 first ICSI-cycles.

We observed an association between increasing times of the pronuclear fading-first cleavage interval (t2-tPNf) and the rates of trichotomous/direct unequal cleavage at the first (DUC-1) and second (DUC-2) mitotic cycle. Moreover, we observed a reduced blastulation rate. No significant associations were observed between rates of direct unequal cleavage at the third mitotic cycle (DUC-3) and top-quality blastocysts, euploidy, and live births. To evaluate whether the interval t2-tPNf could have a predictive value for the onset of DUC-1 and DUC-2, ROC curve analyses were performed. The area under the curve values obtained for DUC-1 showed a significant prediction accuracy. The best cut-offs to identify zygotes with a high risk of DUC-1 and DUC-2 occurrence were t2-tPNf > 2.78 (hours) and t2-tPNf > 2.50 (hours), respectively.

Delay in the short interval between PNBD and the first cytokinesis result in trichotomous cleavage and early developmental arrest. However, if the embryos reach the blastocyst stage, rates of euploidy and live birth do not appear to be compromised.

To evaluate if morphology and morphokinetics of cleaved embryos affect prenatal and perinatal outcomes.

This retrospective cohort study included 734 single fresh embryo transfer (SET) from ICSI from January 2014 to December 2020 at the Dijon University Hospital. Using time-lapse technology, embryos were defined as TOP or non-TOP according to morphological/morphokinetic criteria. Linear regression, adjusted for maternal factors, explored the association between cleaved-embryo quality and implantation rate, miscarriage rate, live birth rate, term, birthweight, and birthweight Z-score.

Multivariate analyses showed that non-implanted embryos were significantly more often out of the interquartile range than implanted embryos for cell events t2 (p = 0.019), t3 (p = 0.038), t4 (p = 0.013), ECC1 (p = 0.047), and ECC2 (p = 0.001). After implantation, the morphokinetics was significantly different for non-TOP embryos ending in miscarriage or live birth. A significant high risk of miscarriage was found for embryos with a short or long ECC1 (3.27 [95% CI: 1.44;8.37]. There was no significant difference in birthweight, birthweight Z-score, or term between TOP and non-TOP embryos. An association with increased birthweight was observed in multivariate analysis for pronuclei appearance (tPNa) (p = 0.001out of the range. Late tPNa was associated with a higher birthweight Z-score (p < 0.0001) while late pronuclei disappearance (tPNf) was associated with a lower Z-score (p = 0.006).

Morphological and morphokinetic parameters significantly influence implantation rates and the likelihood of a live birth from embryos with non-optimal morphological features. Birthweight Z-scores were significantly higher with late tPNa and lower with late tPNf.

Is it possible to predict blastocyst quality, embryo chromosomal ploidy, and clinical pregnancy outcome after single embryo transfer from embryo developmental morphokinetic parameters?

The morphokinetic parameters of 1011 blastocysts from 227 patients undergoing preimplantation genetic testing were examined. Correlations between the morphokinetic parameters and the quality of blastocysts, chromosomal ploidy, and clinical pregnancy outcomes following the transfer of single blastocysts were retrospectively analyzed.

The morphokinetic parameters of embryos in the high-quality blastocyst group were significantly shorter than those in the low-quality blastocyst group (p < 0.05). In contrast, the CC2 time was significantly prolonged (p < 0.05). On chromosomal analysis of biopsy blastocysts nourished by trophectoderm cells, in comparison to euploid embryos, aneuploid embryos exhibited significant extensions in tPNa, S3, tSC, tM, tSB, and tB (p < 0.05), with a simultaneous significant reduction in CC2 time (p < 0.05). After adjusting for age and body mass index through logistic regression analysis, late morphokinetic parameters, namely tM (OR 0.96; 95% CI 0.93-0.99), tSB (OR 0.94; 95% CI 0.90-0.97), and tB (OR 0.93; 95% CI 0.90-0.97), emerged as independent risk factors influencing the development of embryos into high-quality blastocysts. S3 (< 12.01 h), t8 (< 62.48 h), and tPB2 (< 3.36 h) were potential predictors of a successful clinical pregnancy after blastocyst transfer.

Morphokinetic parameters showed correlations with blastocyst quality, chromosomal status, and clinical pregnancy outcomes post-transfer, making them effective predictors for clinical results. Embryos with relatively rapid development tended to exhibit better blastocyst quality, chromosomal ploidy, and improved clinical pregnancy outcomes. The late morphokinetic parameter, S3, demonstrated a strong predictive effect on blastocyst quality, chromosomal ploidy, and clinical pregnancy outcomes.

C-X-C motif chemokine ligand 12 (CXCL12; Stromal Cell-Derived Factor 1 [SDF-1]), most notably known for its role in embryogenesis and hematopoiesis, has been implicated in tumor pathophysiology and neovascularization. However, its cell-specific role and mechanism of action have not been well characterized. Previous work by our group has demonstrated that hypoxia-inducible factor (HIF)-1 modulates downstream CXCL12 expression following ischemic tissue injury. By utilizing a conditional CXCL12 knockout murine model, we demonstrate that endothelial-specific deletion of CXCL12 (eKO) modulates ischemic tissue survival, altering tissue repair and tumor progression without affecting embryogenesis and morphogenesis. Loss of endothelial-specific CXCL12 disrupts critical endothelial-fibroblast crosstalk necessary for stromal growth and vascularization. Using murine parabiosis with novel transcriptomic technologies, we demonstrate that endothelial-specific CXCL12 signaling results in downstream recruitment of non-inflammatory circulating cells, defined by neovascularization modulating genes. These findings indicate an essential role for endothelial-specific CXCL12 expression during the neovascular response in tissue injury and tumor progression.

Infertility is a highly prevalent disease affecting 1 in 6 couples worldwide. While there are numerous treatment options available to treat infertility, in vitro fertilization (IVF) is the most successful therapy available. IVF traditionally involves superovulation of the ovaries followed by ultrasound (US)-guided oocyte retrieval, fertilization of oocytes in vitro, and culture of resultant embryos. Embryo(s) are then either transferred into the uterus or cryopreserved. There have been tremendous advances in each step of this process over the past 40 years, however, much work still remains to be done. This review discusses the history and current techniques involved in oocyte fertilization and embryo culture. Highlighted areas include conventional insemination and intracytoplasmic sperm injection (ICSI), embryo culture, and laboratory assessment. Areas of past and present research including time-lapse imaging and in vitro maturation are also discussed.

Currently, assisted reproduction clinics employ various sperm selection techniques to identify the best sperm for fertilization. However, these techniques may not assess crucial sperm traits that can substantially impact embryonic quality. To address this, we propose analyzing diverse histone modifications as potential markers of sperm functionality and success in assisted reproduction techniques.

Cross-sectional pilot study including infertile male patients attending an infertility clinic in CABA, Argentina between April and August 2019 was performed. We used immunofluorescence techniques to evaluate post-translational modifications of histones in sperm and established correlations with in vitro fertilization outcome and embryo quality.

Our findings indicate a negative correlation between H3K4me3 and H3K4me2 marks and fertilization rate and showed a positive correlation of this parameter with H3K9me mark. In addition, there was a positive correlation between H3K27me3 and good embryo quality.

This pilot study proposes a non-invasive strategy to predict embryo quality by analyzing spermatozoa prior to fertilization. The assessment of histone post-translational modifications in sperm samples could provide useful information for the recognition of epigenetic marks that could predict the health of the embryo of an assisted fertilization treatment.

Septins are a family of cytokinesis-related proteins involved in regulating cytoskeletal design, cell morphology, and tissue morphogenesis. Apart from cytokinesis, as a fourth component of cytoskeleton, septins aid in forming scaffolds, vesicle sorting and membrane stability. They are also known to be involved in the regulation of intracellular calcium (Ca2+) via the STIM/Orai complex. Infertility affects ~ 15% of couples globally, while male infertility affects ~ 7% of men. Global pregnancy and live birth rates following fertility treatment remain relatively low, while there has been an observable decline in male fertility parameters over the past 60 years. Low fertility treatment success can be attributed to poor embryonic development, poor sperm parameters and fertilisation defects. While studies from the past few years have provided evidence for the role of septins in fertility related processes, the functional role of septins and its related complexes in cellular processes such as oocyte activation, fertilization, and sperm maturation are not completely understood. This review summarizes the available knowledge on the role of septins in spermatogenesis and oocyte activation via Ca2+ regulation, and cytoskeletal dynamics throughout pre-implantation embryonic development. We aim to identify the currently less known mechanisms by which septins regulate these immensely important mechanisms with a view of identifying areas of investigation that would benefit our understanding of cell and reproductive biology, but also provide potential avenues to improve current methods of fertility treatment.

The association between embryo morphokinetics and its developmental competence is well documented. For instance, early cleaved embryos are more competent in developing to blastocysts, whereas the proportion of abnormally cleaved embryos that further developed to blastocysts is low. Numerous factors, such as the parental age, lifestyle, health, and smoking habits have been reported to affect the embryo morphokinetics and, consequently, its development. However, less is known about the effect of environmental stressors on embryo morphokinetics. The current review discusses the effect of the most concerning environmental stressors on embryo morphokinetics. These stresses include heat stress and human-made chemicals such as phthalates (e.g., bis-(2-ethylhexyl phthalate, dibutyl phthalate, dimethyl phthalate, and their primary metabolites), herbicides (e.g., diaminochlorotriazine, the primary metabolite of atrazine), pharmaceutical compounds (e.g., carbamazepine, nocodazole) and pro-oxidant agents (cumene hydroperoxide, Triton X-100), as well as naturally occurring toxins such as mycotoxin (e.g., aflatoxin B1 and its metabolite, and ochratoxin A). In addition, this review discusses the effect of ionizing or non-ionizing radiation and viral infections (e.g., SARS-CoV-2, papillomavirus). Finally, it points out some potential mechanisms that underlie the impairment of embryo morphokinetics, and it suggests protective compounds, mainly the supplementation of antioxidants to improve the morphokinetics, and consequently, the embryo developmental competence.

Infertility is a global reproductive health burden. Assisted reproductive technologies (ARTs) have been widely used to help patients become pregnant. Few embryos develop to the blastocyst stage with ARTs, leading to relatively low live birth rates. Protein modifications play crucial roles in nearly every aspect of cell biology, including reproductive processes. The aim of this study was to explore the characteristics of protein modifications during embryonic development.

Proteomic data from humans and mice were acquired from the integrated proteome resources (iProX) of ProteomeXchange (PXD024267) and a tandem mass tag (TMT)-mass spectrometry dataset. Gene ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were applied for functional annotation. Protein-protein interactions (PPIs) of the modification-related genes were revealed by the STRING database. Modified proteins during mouse embryogenesis were visualized through heatmaps of hierarchically clustering using k-means.

We identified modification-related proteins in human embryo development and characterized them through heatmaps, GO analysis, KEGG analysis, and PPI network analysis. We found that the 4-cell stage to the 8-cell stage might be the demarcation period for modification-related protein expression patterns during embryo development. Using quantitative mass spectrometry, we elucidated the methylation, acetylation, and ubiquitination events that occur during mouse embryogenesis to validate our findings in human embryonic development to some extent.

The results of our study suggest that the posttranslational modifications (PTMs) of human preimplantation embryos might exhibit the same trends as those in mice to exert synergistic and fine-tuned regulatory effects during embryonic development.

Time-lapse microscopy for embryos is a non-invasive technology used to characterize early embryo development. This study employs time-lapse microscopy and machine learning to elucidate changes in embryonic growth kinetics with maternal aging. We analyzed morphokinetic parameters of embryos from young and aged C57BL6/NJ mice via continuous imaging. Our findings show that aged embryos accelerated through cleavage stages (from 5-cells) to morula compared to younger counterparts, with no significant differences observed in later stages of blastulation. Unsupervised machine learning identified two distinct clusters comprising of embryos from aged or young donors. Moreover, in supervised learning, the extreme gradient boosting algorithm successfully predicted the age-related phenotype with 0.78 accuracy, 0.81 precision, and 0.83 recall following hyperparameter tuning. These results highlight two main scientific insights: maternal aging affects embryonic development pace, and artificial intelligence can differentiate between embryos from aged and young maternal mice by a non-invasive approach. Thus, machine learning can be used to identify morphokinetics phenotypes for further studies. This study has potential for future applications in selecting human embryos for embryo transfer, without or in complement with preimplantation genetic testing.

Are sperm phospholipase C zeta (PLCζ) profiles linked to the quality of embryogenesis and pregnancy?

Sperm PLCζ levels in both mouse and humans correlate with measures of ideal embryogenesis whereby minimal levels seem to be required to result in successful pregnancy.

While causative factors underlying male infertility are multivariable, cases are increasingly associated with the efficacy of oocyte activation, which in mammals occurs in response to specific profiles of calcium (Ca2+) oscillations driven by sperm-specific PLCζ. Although sperm PLCζ abrogation is extensively linked with human male infertility where oocyte activation is deficient, less is clear as to whether sperm PLCζ levels or localization underlies cases of defective embryogenesis and failed pregnancy following fertility treatment.

A cohort of 54 couples undergoing fertility treatment were recruited at the assisted reproductive technology laboratory at the King Faisal Hospital and Research Centre, Riyadh, Kingdom of Saudi Arabia. The recruitment criteria for males was a minimum sperm concentration of 5×106 sperm/ml, while all female patients had to have at least five oocytes. Sperm PLCζ analysis was performed in research laboratories, while semen assessments were performed, and time-lapse morphokinetic data were obtained, in the fertility clinic as part of routine treatment. The CRISPR/Cas9 system was concurrently used to induce indels and single-nucleotide mutations within the Plcζ gene to generate strains of Plcζ mutant mice. Sperm PLCζ was evaluated using immunofluorescence and immunoblotting with an antibody of confirmed consistent specificity against PLCζ.

We evaluated PLCζ profiles in sperm samples from 54 human couples undergoing fertility treatment in the context of time-lapse morphokinetic analysis of resultant embryos, correlating such profiles to pregnancy status. Concurrently, we generated two strains of mutant Plcζ mice using CRISPR/Cas9, and performed IVF with wild type (WT) oocytes and using WT or mutant Plcζ sperm to generate embryos. We also assessed PLCζ status in WT and mutant mice sperm in the context of time-lapse morphokinetic analysis and breeding outcomes.

A significant (P ≤ 0.05) positive relationship was observed between both PLCζ relative fluorescence and relative density with the times taken for both the second cell division (CC2) (r = 0.26 and r = 0.43, respectively) and the third cell division (S2) (r = 0.26). Examination of localization patterns also indicated significant correlations between the presence or absence of sperm PLCζ and CC2 (r = 0.27 and r = -0.27, respectively; P ≤ 0.025). Human sperm PLCζ levels were at their highest in the ideal times of CC2 (8-12 h) compared to time ranges outside the ideal timeframe (<8 and >12 h) where levels of human sperm PLCζ were lower. Following assignment of PLCζ level thresholds, quantification revealed a significantly higher (P ≤ 0.05) rate of successful pregnancy in values larger than the assigned cut-off for both relative fluorescence (19% vs 40%, respectively) and relative density (8% vs 54%, respectively). Immunoblotting indicated a single band for PLCζ at 74 kDa in sperm from WT mice, while a single band was also observed in sperm from heterozygous of Plcζ mutant mouse sperm, but at a diminished intensity. Immunofluorescent analysis indicated the previously reported (Kashir et al., 2021) fluorescence patterns in WT sperm, while sperm from Plcζ mutant mice exhibited a significantly diminished and dispersed pattern at the acrosomal region of the sperm head. Breeding experiments indicated a significantly reduced litter size of mutant Plcζ male mice compared to WT mice, while IVF-generated embryos using sperm from mutant Plcζ mice exhibited high rates of polyspermy, and resulted in significantly reduced numbers of these embryos reaching developmental milestones.

The human population examined was relatively small, and should be expanded to examine a larger multi-centre cohort. Infertility conditions are often multivariable, and it was not possible to evaluate all these in human patients. However, our mutant Plcζ mouse experiments do suggest that PLCζ plays a significant role in early embryo development.

We found that minimal levels of PLCζ within a specific range were required for optimal early embryogenesis, correlating with increased pregnancy. Levels of sperm PLCζ below specific thresholds were associated with ineffective embryogenesis and lower pregnancy rates, despite eliciting successful fertilization in both mice and humans. To our knowledge, this represents the first time that PLCζ levels in sperm have been correlated to prognostic measures of embryogenic efficacy and pregnancy rates in humans. Our data suggest for the first time that the clinical utilization of PLCζ may stand to benefit not just a specific population of male infertility where oocyte activation is completely deficient (wherein PLCζ is completely defective/abrogated), but also perhaps the larger population of couples seeking fertility treatment.

J.K. is supported by a faculty start up grant awarded by Khalifa University (FSU-2023-015). This study was also supported by a Healthcare Research Fellowship Award (HF-14-16) from Health and Care Research Wales (HCRW) to J.K., alongside a National Science, Technology, and Innovation plan (NSTIP) project grant (15-MED4186-20) awarded by the King Abdulaziz City for Science and Technology (KACST) for J.K. and A.M.A. The authors declare no conflicts of interest.

N/A.

What is the utilization rate of embryos that exert inadequate zygote cleavage into three daughter cells?

This study used a retrospective dataset from a single IVF Unit. A total of 3,060 embryos from 1,811 fresh IVF cycles were analyzed. The cleavage pattern, morphokinetics, and outcome were recorded. Only 2pn embryos, fertilized by ejaculated sperm, and cultured in a time-lapse system for at least 5 days were included. We generated three study groups according to the embryo's cleavage pattern: (I) Control, normal cleavage (n = 551); (II) fast cleavage, zygote to three cells within 5 h (n = 1,587); and (III) instant direct tripolar cleavage (IDC) from zygote to three cells (n = 922).

The rate of usable fast cleavage blastocysts was 108/1,587 (6.81%) and usable control blastocysts was 180/551 (32.67%). The time of PN fading and from fading to first cleavage differed significantly between the three groups. Although the pregnancy rate of control and fast cleavage blastocysts were comparable (40.35% and 42.55%, respectively), the amount of instant direct cleavage embryos that reached blastocyst stage was neglectable (only four embryos out of 922 analyzed IDC embryos) and unsuitable for statistical comparison of pregnancy rates.

Our results indicate the need to culture instant direct cleavage embryos for 5 days, up to the blastocyst stage, and avoid transfer of embryos that are fated to arrest even when their morphological grade on day 3 is acceptable, whereas fast cleavage embryos could be transferred on day 3 when there is no alternative.

Can generative artificial intelligence (AI) models produce high-fidelity images of human blastocysts?

Generative AI models exhibit the capability to generate high-fidelity human blastocyst images, thereby providing substantial training datasets crucial for the development of robust AI models.

The integration of AI into IVF procedures holds the potential to enhance objectivity and automate embryo selection for transfer. However, the effectiveness of AI is limited by data scarcity and ethical concerns related to patient data privacy. Generative adversarial networks (GAN) have emerged as a promising approach to alleviate data limitations by generating synthetic data that closely approximate real images.

Blastocyst images were included as training data from a public dataset of time-lapse microscopy (TLM) videos (n = 136). A style-based GAN was fine-tuned as the generative model.

We curated a total of 972 blastocyst images as training data, where frames were captured within the time window of 110-120 h post-insemination at 1-h intervals from TLM videos. We configured the style-based GAN model with data augmentation (AUG) and pretrained weights (Pretrained-T: with translation equivariance; Pretrained-R: with translation and rotation equivariance) to compare their optimization on image synthesis. We then applied quantitative metrics including Fréchet Inception Distance (FID) and Kernel Inception Distance (KID) to assess the quality and fidelity of the generated images. Subsequently, we evaluated qualitative performance by measuring the intelligence behavior of the model through the visual Turing test. To this end, 60 individuals with diverse backgrounds and expertise in clinical embryology and IVF evaluated the quality of synthetic embryo images.

During the training process, we observed consistent improvement of image quality that was measured by FID and KID scores. Pretrained and AUG + Pretrained initiated with remarkably lower FID and KID values compared to both Baseline and AUG + Baseline models. Following 5000 training iterations, the AUG + Pretrained-R model showed the highest performance of the evaluated five configurations with FID and KID scores of 15.2 and 0.004, respectively. Subsequently, we carried out the visual Turing test, such that IVF embryologists, IVF laboratory technicians, and non-experts evaluated the synthetic blastocyst-stage embryo images and obtained similar performance in specificity with marginal differences in accuracy and sensitivity.

In this study, we primarily focused the training data on blastocyst images as IVF embryos are primarily assessed in blastocyst stage. However, generation of an array of images in different preimplantation stages offers further insights into the development of preimplantation embryos and IVF success. In addition, we resized training images to a resolution of 256 × 256 pixels to moderate the computational costs of training the style-based GAN models. Further research is needed to involve a more extensive and diverse dataset from the formation of the zygote to the blastocyst stage, e.g. video generation, and the use of improved image resolution to facilitate the development of comprehensive AI algorithms and to produce higher-quality images.

Generative AI models hold promising potential in generating high-fidelity human blastocyst images, which allows the development of robust AI models as it can provide sufficient training datasets while safeguarding patient data privacy. Additionally, this may help to produce sufficient embryo imaging training data with different (rare) abnormal features, such as embryonic arrest, tripolar cell division to avoid class imbalances and reach to even datasets. Thus, generative models may offer a compelling opportunity to transform embryo selection procedures and substantially enhance IVF outcomes.

This study was supported by a Horizon 2020 innovation grant (ERIN, grant no. EU952516) and a Horizon Europe grant (NESTOR, grant no. 101120075) of the European Commission to A.S. and M.Z.E., the Estonian Research Council (grant no. PRG1076) to A.S., and the EVA (Erfelijkheid Voortplanting & Aanleg) specialty program (grant no. KP111513) of Maastricht University Medical Centre (MUMC+) to M.Z.E.

Not applicable.

This study aims to construct and validate a nomogram for predicting blastocyst formation in patients with diminished ovarian reserve (DOR) during in vitro fertilization (IVF) procedures. A retrospective analysis was conducted on 445 DOR patients who underwent in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) at the Reproductive Center of Yulin Maternal and Child Health Hospital from January 2019 to January 2023. A total of 1016 embryos were cultured for blastocyst formation, of which 487 were usable blastocysts and 529 did not form usable blastocysts. The embryos were randomly divided into a training set (711 embryos) and a validation set (305 embryos). Relevant factors were initially identified through univariate logistic regression analysis based on the training set, followed by multivariate logistic regression analysis to establish a nomogram model. The prediction model was then calibrated and validated. Multivariate stepwise forward logistic regression analysis showed that female age, normal fertilization status, embryo grade on D2, and embryo grade on D3 were independent predictors of blastocyst formation in DOR patients. The Hosmer-Lemeshow test indicated no statistical difference between the predicted probabilities of blastocyst formation and actual blastocyst formation (P > 0.05). These results suggest that female age, normal fertilization status, embryo grade on D2, and embryo grade on D3 are independent predictors of blastocyst formation in DOR patients. The clinical prediction nomogram constructed from these factors has good predictive value and clinical utility and can provide a basis for clinical prognosis, intervention, and the formulation of individualized medical plans.

Ovarian stimulation protocols are widely used to collect oocytes in assisted reproductive technologies (ARTs). Although the influence of ovarian stimulation on embryo quality has been described, this issue remains controversial. Here, we analyzed the influence of ovarian stimulation on developmental speed and chromosome segregation using live cell imaging. Female mice at the proestrus stage were separated by the appearance of the vagina as the non-stimulation (-) group, and other mice were administered pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) as the stimulation (+) groups. The cumulus-oocyte complexes from both groups were inseminated with sperm suspensions from the same male mice. Fertilization rates and developmental capacities were examined, and the developmental speed and frequency of chromosome segregation errors were measured by live-cell imaging using a Histone H2B-mCherry probe. The number of fertilized oocytes obtained was 1.4-fold more frequent in the stimulation (+) group. The developmental rate and chromosome stability did not differ between the groups. Image analysis showed that the mean speed of development in the stimulation (+) group was slightly higher than that in the non-stimulation (-) group. This increase in speed seemed to arise from the slight shortening of the 2- and 4-cell stages and third division lengths and consequent synchronization of cleavage timing in each embryo, not from the emergence of an extremely rapidly developing subpopulation of embryos. In conclusion, ovarian stimulation does not necessarily affect embryo quality but rather increases the chances of obtaining high-quality oocytes in mice.

Is there an association between morphokinetic variables of meiotic maturation and the severity of aneuploidy following in vitro maturation (IVM) in the mouse?

The severity of meiotic aneuploidy correlates with an extended time to first polar body extrusion (tPB1) and duration of meiosis I (dMI).

Morphokinetic variables measured using time-lapse technology allow for the non-invasive evaluation of preimplantation embryo development within clinical assisted reproductive technology (ART). We recently applied this technology to monitor meiotic progression during IVM of mouse gametes. Whether there is a relationship between morphokinetic variables of meiotic progression and aneuploidy in the resulting egg has not been systematically examined at the resolution of specific chromosomes. Next-generation sequencing (NGS) is a robust clinical tool for determining aneuploidy status and has been reverse-translated in mouse blastocysts and oocytes. Therefore, we harnessed the technologies of time-lapse imaging and NGS to determine the relationship between the morphokinetics of meiotic progression and egg aneuploidy.

Cumulus-oocyte complexes were collected from large antral follicles from hyperstimulated CD-1 mice. Cumulus cells were removed, and spontaneous IVM was performed in the absence or presence of two doses of Nocodazole (25 or 50 nM) to induce a spectrum of spindle abnormalities and chromosome segregation errors during oocyte meiosis. Comprehensive chromosome screening was then performed in the resulting eggs, and morphokinetic variables and ploidy status were compared across experimental groups (control, n = 11; 25 nM Nocodazole, n = 13; 50 nM Nocodazole, n = 23).

We monitored IVM in mouse oocytes using time-lapse microscopy for 16 h, and time to germinal vesicle breakdown (tGVBD), tPB1, and dMI were analyzed. Following IVM, comprehensive chromosome screening was performed on the eggs and their matched first polar bodies via adaptation of an NGS-based preimplantation genetic testing for aneuploidy (PGT-A) assay. Bioinformatics analysis was performed to align reads to the mouse genome and determine copy number-based predictions of aneuploidy. The concordance of each polar body-egg pair (reciprocal errors) was used to validate the results. Ploidy status was categorized as euploid, 1-3 chromosomal segregation errors, or ≥4 chromosomal segregation errors. Additionally, aneuploidy due to premature separation of sister chromatids (PSSC) versus non-disjunction (NDJ) was distinguished.

We applied and validated state-of-the-art NGS technology to screen aneuploidy in individual mouse eggs and matched polar bodies at the chromosome-specific level. By performing IVM in the presence of different doses of Nocodazole, we induced a range of aneuploidy. No aneuploidy was observed in the absence of Nocodazole (0/11), whereas IVM in the presence of 25 and 50 nM Nocodazole resulted in an aneuploidy incidence of 7.69% (1/13) and 82.61% (19/23), respectively. Of the aneuploid eggs, 5% (1/20) was due to PSSC, 65% (13/20) to NDJ, and the remainder to a combination of both. There was no relationship between ploidy status and tGVBD, but tPB1 and the dMI were both significantly prolonged in eggs with reciprocal aneuploidy events compared to the euploid eggs, and this scaled with the severity of aneuploidy. Eggs with ≥4 aneuploid chromosomes had the longest tPB1 and dMI (P < 0.0001), whereas eggs with one to three aneuploid chromosomes exhibited intermediate lengths of time (P < 0.0001).

N/A.

We used Nocodazole in this study to disrupt the meiotic spindle and induce aneuploidy in mouse oocytes. Whether the association between morphokinetic variables of meiotic progression and the severity of aneuploidy occurs with other compounds that induce chromosome segregation errors remain to be investigated. In addition, unlike mouse oocytes, human IVM requires the presence of cumulus cells, which precludes visualization of morphokinetic variables of meiotic progression. Thus, our study may have limited direct clinical translatability.

We validated NGS in mouse eggs to detect aneuploidy at a chromosome-specific resolution which greatly improves the utility of the mouse model. With a tractable and validated model system for characterizing meiotic aneuploidy, investigations into the molecular mechanisms and factors which may influence aneuploidy can be further elaborated. Time-lapse analyses of morphokinetic variables of meiotic progression may be a useful non-invasive predictor of aneuploidy severity.

This work was supported by the Bill & Melinda Gates Foundation (INV-003385). Under the grant conditions of the Foundation, a Creative Commons Attribution 4.0 Generic License has already been assigned to the Author Accepted Manuscript version that might arise from this submission. The authors have no conflict of interest to disclose.

Preimplantation genetic testing (PGT) has become a common supplementary diagnοstic/testing tοol for in vitro fertilization (ΙVF) cycles due to a significant increase in cases of PGT fοr mοnogenic cοnditions (ΡGT-M) and de novο aneuplοidies (ΡGT-A) over the last ten years. This tendency is mostly attributable to the advancement and application of novel cytogenetic and molecular techniques in clinical practice that are capable of providing an efficient evaluation of the embryonic chromosomal complement and leading to better IVF/ICSI results. Although PGT is widely used, it requires invasive biopsy of the blastocyst, which may harm the embryo. Non-invasive approaches, like cell-free DNA (cfDNA) testing, have lower risks but have drawbacks in consistency and sensitivity. This review discusses new developments and opportunities in the field of preimplantation genetic testing, enhancing the overall effectiveness and accessibility of preimplantation testing in the framework of developments in genomic sequencing, bioinformatics, and the integration of artificial intelligence in the interpretation of genetic data.

Artificial Intelligence entails the application of computer algorithms to the huge and heterogeneous amount of morphodynamic data produced by Time-Lapse Technology. In this context, Machine Learning (ML) methods were developed in order to assist embryologists with automatized and objective predictive models able to standardize human embryo assessment. In this study, we aimed at developing a novel ML-based strategy to identify relevant patterns associated with the prediction of blastocyst development stage on day 5.

We retrospectively analysed the morphokinetics of 575 embryos obtained from 80 women who underwent IVF at our Unit. Embryo morphokinetics was registered using the Geri plus® time-lapse system. Overall, 30 clinical, morphological and morphokinetic variables related to women and embryos were recorded and combined. Some embryos reached the expanded blastocyst stage on day 5 (BL Group, n = 210), some others did not (nBL Group, n = 365).

The novel EmbryoMLSelection framework was developed following four-steps: Feature Selection, Rules Extraction, Rules Selection and Rules Evaluation. Six rules composed by a combination of 8 variables were finally selected, and provided a predictive power described by an AUC of 0.84 and an accuracy of 81%.

We provided herein a new feature-signature able to identify with an high performance embryos with the best developmental competence to reach the expanded blastocyst stage on day 5. Clear and clinically relevant cut-offs were identified for each considered variable, providing an objective tool for early embryo developmental assessment.

There are many instances of hollow-structure morphogenesis in the development of tissues. Thus, the fabrication of hollow structures in a simple, high-throughput and homogeneous manner with proper natural biomaterial combination is valuable for developmental studies and tissue engineering, while it is a significant challenge in biofabrication field. We present a novel method for the fabrication of a hollow cell module using a coaxial co-flow capillary microfluidic device. Sacrificial gelatin laden with cells in the inner layer and GelMa in the outer layer are used via a coaxial co-flow capillary microfluidic device to produce homogenous micro-beads. The overall and core sizes of core-shell microbeads were well controlled. When using human vein vascular endothelial cells to demonstrate how cells line the inner surface of core-shell beads, as the core liquifies, a hollow cell ball with asymmetric features is fabricated. After release from the GelMa shell, individual cell balls are obtained and deformed cell balls can self-recover. This platform paves way for complex hollow tissue modeling in vitro, and further modulation of matrix stiffness, curvature and biochemical composition to mimic in vivo microenvironments.

The study of pig preimplantation embryo development has several potential uses: from agriculture to the production of medically relevant genetically modified organisms and from rare breed conservation to acting as a physiologically relevant model for progressing human and other (e.g., endangered) species' in vitro fertilisation technology. Despite this, barriers to the widespread adoption of pig embryo in vitro production include lipid-laden cells that are hard to visualise, slow adoption of contemporary technologies such as the use of time-lapse incubators or artificial intelligence, poor blastulation and high polyspermy rates. Here, we employ a commercially available time-lapse incubator to provide a comprehensive overview of the morphokinetics of pig preimplantation development for the first time. We tested the hypotheses that (a) there are differences in developmental timings between blastulating and non-blastulating embryos and (b) embryo developmental morphokinetic features can be used to predict the likelihood of blastulation. The abattoir-derived oocytes fertilised by commercial extended semen produced presumptive zygotes were split into two groups: cavitating/blastulating 144 h post gamete co-incubation and those that were not. The blastulating group reached the 2-cell and morula stages significantly earlier, and the time taken to reach the 2-cell stage was identified to be a predictive marker for blastocyst formation. Reverse cleavage was also associated with poor blastulation. These data demonstrate the potential of morphokinetic analysis in automating and upscaling pig in vitro production through effective embryo selection.

The selection of embryos is a key for the success of in vitro fertilization (IVF). However, automatic quality assessment on human IVF embryos with optical microscope images is still challenging. In this study, we developed a clinical consensus-compliant deep learning approach, named Esava (Embryo Segmentation and Viability Assessment), to quantitatively evaluate the development of IVF embryos using optical microscope images. In total 551 optical microscope images of human IVF embryos of day-2 to day-3 were collected, preprocessed, and annotated. Using the Faster R-CNN model as baseline, our Esava model was constructed, refined, trained, and validated for precise and robust blastomere detection. A novel algorithm Crowd-NMS was proposed and employed in Esava to enhance the object detection and to precisely quantify the embryonic cells and their size uniformity. Additionally, an innovative GrabCut-based unsupervised module was integrated for the segmentation of blastomeres and embryos. Independently tested on 94 embryo images for blastomere detection, Esava obtained the high rates of 0.9940, 0.9121, and 0.9531 for precision, recall, and mAP respectively, and gained significant advances compared with previous computational methods. Intraclass correlation coefficients indicated the consistency between Esava and three experienced embryologists. Another test on 51 extra images demonstrated that Esava surpassed other tools significantly, achieving the highest average precision 0.9025. Moreover, it also accurately identified the borders of blastomeres with mIoU over 0.88 on the independent testing dataset. Esava is compliant with the Istanbul clinical consensus and compatible to senior embryologists. Taken together, Esava improves the accuracy and efficiency of embryonic development assessment with optical microscope images.

The goal of assisted reproduction is for a couple treated with IVF techniques to end the treatment by giving birth to a healthy baby. A neccessary presumption for success is the identification of the best embryo with high implantation and developmental potential. One option is to select an euploid embryo by invasive preimplantaion genetic testing for aneuploidy (PGT-A) or it is possible to select the best embryo by non-invasive time-lapse monitoring (TLM), specifically based on morphokinetic parameters and morphological markers that are able to identify an embryo with high developmental potential.

The study involved a total of 1060 embryos (585 euploid and 475 aneuploid embryos after PGT-A) with good morphology from 329 patients in the period 01/2016-10/2021. All embryos were cultured in a time-lapse incubator, trophectoderm (TE) cells biopsies for PGT-A examination were performed on day 5 (D5) or day 6 (D6) of culture. During the study period, 225 frozen embryo transfers (FET) of one euploid embryo were performed. Based on the treatment outcome, the embryos were divided into 2 groups - euploid embryos, which led to the birth of a healthy child, and euploid embryos that did not show fetal heartbeat (FHB) after FET.

Based on the statistical analysis of the embryos without implantation and the embryos with live birth, it is clear that the morphokinetic parameters t5 (time of division into 5 cells) and tSB (time of start of blastulation) are significantly different.

The results suggest that of the morphokinetic parameters tSB and t5 are predictive indicators for selecting an embryo with high developmental potential and with a high probability of achieving the birth of a healthy child.

Time-lapse technology (TLT) has gained widespread adoption worldwide. In addition to facilitating the undisturbed culture of embryos, TLT offers the unique capability of continuously monitoring embryos to detect spatiotemporal changes. Although these observed phenomena play a role in optimal embryo selection/deselection, the clinical advantages of introducing TLT remain unclear. However, manual annotation of embryo perturbation could facilitate a comprehensive assessment of developmental competence. This process requires a thorough understanding of embryo observation and the biological significance associated with developmental dogma and variation. This review elucidates the typical behavior and variation of each phenomenon, exploring their clinical significance and research perspectives.

The MEDLINE database was searched using PubMed for peer-reviewed English-language original articles concerning human embryo development.

TLT allows the observation of consecutive changes in embryo morphology, serving as potential biomarkers for embryo assessment. In assisted reproductive technology laboratories, several phenomena have not revealed their mechanism, posing difficulties such as fertilization deficiency and morula arrest.

A profound understanding of the biological mechanisms and significance of each phenomenon is crucial. Further collaborative efforts between the clinical and molecular fields following translational studies are required to advance embryonic outcomes and assessment.

The Association for the Study of Reproductive Biology (ASEBIR) Interest Group in Embryology (in Spanish 'Grupo de Interés de Embriología') reviewed key morphokinetic parameters to assess the contribution of time-lapse technology (TLT) to the ASEBIR grading system. Embryo grading based on morphological characteristics is the most widely used method in human assisted reproduction laboratories. The introduction and implementation of TLT has provided a large amount of information that can be used as a complementary tool for morphological embryo evaluation and selection. As part of IVF treatments, embryologists grade embryos to decide which embryos to transfer or freeze. At the present, the embryo grading system developed by ASEBIR does not consider dynamic events observed through TLT. Laboratories that are using TLT consider those parameters as complementary data for embryo selection. The aim of this review was to evaluate review time-specific morphological changes during embryo development that are not included in the ASEBIR scoring system, and to consider them as candidates to add to the scoring system.

Embryo morphokinetic analysis through time-lapse embryo imaging is envisioned as a method to improve selection of developmentally competent embryos. Morphokinetic analysis could be utilized to evaluate the effects of experimental manipulation on pre-implantation embryo development. The objectives of this study were to establish a normative morphokinetic database for in vitro fertilized rhesus macaque embryos and to assess the impact of atypical initial cleavage patterns on subsequent embryo development and formation of embryo outgrowths. The cleavage pattern and the timing of embryo developmental events were annotated retrospectively for unmanipulated in vitro fertilized rhesus macaque blastocysts produced over four breeding seasons. Approximately 50% of the blastocysts analyzed had an abnormal early cleavage event. The time to the initiation of embryo compaction and the time to completion of hatching was significantly delayed in blastocysts with an abnormal early cleavage event compared to blastocysts that had cleaved normally. Embryo hatching, attachment to an extracellular matrix, and growth during the implantation stage in vitro was not impacted by the initial cleavage pattern. These data establish normative morphokinetic parameters for in vitro fertilized rhesus macaque embryos and suggest that cleavage anomalies may not impact embryo implantation rates following embryo transfer.

This study evaluated the relationship between cytoplasmic granulation patterns and the developmental potential of mature sibling oocytes.

Data from 54 cycles of preimplantation genetic tests for structural rearrangement from July 2019 to June 2022 were analyzed. In total, 564 embryos were cultured using a time-lapse system. Sibling oocytes were divided into four groups based on cytoplasmic granulation patterns: fine granulation (FG) group (n = 177), central granulation (CG) group (n = 183), dispersed granulation (DG) group (n = 161), and uneven granulation (UG) group (n = 43). The CG group was further divided into three groups (grades I, II, and III) based on the tertile of the ratio of central granular distribution area to oocyte area. Fertilization rate, embryo morphokinetics, chromosomal ploidy, and clinical outcomes of the groups were compared.

No significant differences were observed in morphokinetic parameters, fertilization rate, embryo production, blastocyst formation, and aneuploidy rates among the different cytoplasmic-granulation pattern groups. However, embryos derived from CG oocytes showed significantly higher aneuploidy rates in grade III compared to grade I (86.21% vs 61.54%, P = 0.036) or grade II (86.21% vs 56.00%, P = 0.013). Thirty embryos were transferred to the uteri of female patients and the clinical pregnancy and live birth rates did not significantly differ among groups.

Cytoplasmic granulation patterns may not affect embryo fertilization, development speed, and aneuploidy rates. However, a higher grade of CG may be associated with increased aneuploidy rates. Larger sample sizes are required to explore the impact of oocyte cytoplasmic granulation patterns on embryo implantation potential.

Can embryos harbouring cell exclusion and their reproductive outcomes be classified based on morphokinetic profiles?

A total of 469 time-lapse videos of embryos transferred between 2013 and 2019 from a single clinic were analysed. Videos were assessed and grouped according to the presence or absence of one or more excluded cells before compaction. Cell division timings, intervals between subsequent cell divisions and dynamic intervals were analysed to determine the morphokinetic profiles of embryos with cell exclusion (CE+), compared with fully compacted embryos without cell exclusion or extrusion (CE-).

Transfer of CE+ embryos resulted in lower proportions of fetal heartbeat (FHB) and live birth compared with CE- embryos (both, P < 0.001). CE+ embryos were associated with delays in t2 (P = 0.030), t6 (P = 0.018), t7 (P < 0.001), t8 (P = 0.001), tSC (P < 0.001) and tM (P < 0.001). Earlier timings for t3 (P = 0.014) and t5 (P < 0.001) were positively associated with CE+; CE+ embryos indicated prolonged S2, S3, ECC3, cc2 and cc4. Logistic regression analysis revealed that t5, tM, S2 and ECC3 were the strongest predictive indicators of cell exclusion. Timings for S2 and ECC3 were useful in identifying increased odds of FHB when a cell exclusion event was present.

Embryos harbouring cell exclusion indicated altered morphokinetic profiles. Their overall lower reproductive success was associated with two morphokinetic parameters. Morphokinetic profiles could be used as adjunct indicators for reproductive success during cycles producing few, low-quality embryos. This may allow more objective identification of cell exclusion and refinement of embryo ranking procedures before transfer.

Previous studies looked into the connections between pregnancy and the Zona Pellucida (ZP) thickness and Zona Pellucida Thickness Variation (ZPTV), as well as the embryo's radius, circumference, perimeter and global symmetry. However, no research has linked embryo implantation and pregnancy to the percentage of ZP thinning, the reduction in ooplasm volume, and the increase in perivitelline space (PVS) volume. Our objective is to correlate the percentage of ZP thinning, the percentage of ooplasm volume shrinkage and the percentage of PVS increase to the implantation. These data will be used for embryo selection as well as it can be put into a software that will assist embryo selection.

Retrospective study included 281 patients, all of them had 2 embryos transferred, 149 patients got pregnant with two gestation sacs and 132 patients did not get pregnant. All of the transferred embryos had the ZP thickness measured several times from time of ICSI till Embryo Transfer (ET), the ooplasm volume was calculated from time of ICSI till two Pronuclei (2PN) fading and the PVS was calculated from the ICSI time till the 2PN fading.

The first characteristic is the change in the average ZP thickness that decreased by 32.7% + 5.3% at 70 h for the implanted embryos (Group 1) versus 23.6% + 4.8% for non-implanted embryos (Group 2) p = 0.000. The second characteristic is the average reduction in the volume of the ooplasm which is 20.5% + 4.3% in Group 1 versus 15.1% + 5.2% in Group 2, p = 0.000. The third characteristic is the increase in the volume of the PVS which was 38.1% + 7.6% in Group 1 versus 31.6% + 9.7% in Group 2 p = 0.000.

The implanted embryos showed higher percent of ZP thinning, higher percent of ooplasm reduction and higher percent of PVS increase.

Cultivation and selection of high-quality human embryos are critical for the success of in vitro fertilization-embryo transfer. Time-lapse imaging technology (TLI) provides a stable culture environment for embryos, which can continuously observe and record the development process of early embryos, so that doctors can record embryo development time parameters more accurately. In this study, we review the current observation and research on the main embryo dynamics parameters in TLI and discusses their significance and development for embryo development potential. To analysis and summary, the application and research situation of TLI, we searched PubMed, Web of Science, and China National Knowledge Infrastructure, using TLI, embryo dynamics parameters, embryo development potential as Keywords, cited 50 out of the initial 89 selected literatures and summarized. With comparative analysis and research, we found that the embryo dynamic parameters provided by TLI has been intensively studied in clinical empirical and observational research, extensive experimental data verified its effectiveness and advantages in embryo development potential assessment. TLI provides technical support of embryo dynamic parameters, which may become the quantitative indicators for superior embryos and pregnancy prediction as well. Existing studies have shown that certain kinetic parameters provided by TLI culture can predict embryo implantation, but no parameter has been confirmed as the absolute correlation biological indicators yet. In this review we believe that further research is needed to verify these preliminary and sometimes contradictory results, and explore the predictive significance of various embryo kinetic parameters relying on TLI technology for embryo development potential.

The objective of this study was to evaluate the time of blastulation monitored by time-lapse technology to predict in vitro viability of bovine blastocysts. This technology can be a powerful tool for bovine embryos selection with higher implantation capacity and competence. Also, in humans an early blastulation is associated with higher quality and pregnancy rate. Cumulus oocyte complexes (COCs) were matured for 20 to 22 h and then fertilized by co-incubation of COCs and spermatozoa (10,000 sperm per oocyte) for 18 h. Presumptive zygotes were placed individually in microwells, in droplets of commercial culture medium. The Primo Vision TL system (EVO+; Vitrolife) captured digital images of developing embryos every 15 minutes. The time frame from IVF to the start of blastulation (tSB) and to blastocyst development (tB) was recorded. After day 7.5, the blastocysts were in vitro culture for 48 h until day 9.5 after IVF to evaluate post hatching development. In vitro viability was evaluated at day 9.5: those with a diameter greater than 200 μm and a total cell count greater than 180 were classified as viable (value 1), while the rest were classified as non in vitro viable (value 0). The area under the ROC curve (AUC) was estimated to determine the predictive power of in vitro viability through blastulation time. In addition, binary logistic regression analysis was used to generate a mathematical model with morphokinetic variables that allow the best prediction of in vitro viability. In 13 sessions, the blastocyst production rate was 46.2% (96/208). The cut-off time to discriminate early or late blastulation was 149.8 h. The post-hatching development of the embryos with early blastulation was 63.3% (31/49), being statistically superior (p = 0.001) than the late blastulation group 14.9% (7/47). Likewise, the time of blastulation showed an accuracy of 90.8% (p < 0.001) in predicting in vitro viability of bovine blastocysts. In conclusion, the selection of blastocysts based on blastulation time (< 155 h) and blastocyst diameter measured on day 7.5 after IVF (> 180 μm) maximizes the in vitro viability.