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Objective: To study the crosstalk between maternal immune cells and the developing embryo by investigating the immunogenic properties of human blastocyst spent media (SM) on dendritic cells., Methods: In this prospective multicenter experimental study, human preimplantation embryo spent media were collected after blastocyst formation, grouped based on successful or unsuccessful implantation, and analyzed by protein array or used to stimulate monocyte derived dendritic cells (moDC). The immunomodulatory properties of SM on moDC were investigated by analyzing changes in phenotype, cytokine secretion, indoleamine 2,3-dioxygenase (IDO) activity, and ability to activate T cells., Results: A plethora of cytokines and growth factors secreted from preimplantation embryos was detected. Exposure to embryo SM altered the phenotype of moDC in a manner dependent on the implantation outcome. Specifically, SM from non-implanted embryos increased the expression of co-stimulatory molecules and activation markers on moDC. Furthermore, SM treated dendritic cells secreted low levels of cytokines and growth factors and were able to stimulate naïve T cells. Activation of IDO was decreased in moDC after stimulation with SM., Conclusions: Our findings show that human preimplantation embryos secrete an abundance of molecules with the ability to significantly affect and even regulate immune cells in their environment., Competing Interests: Declarations. Ethics approval and consent to participate: The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the ethics committee of the Medical University of Innsbruck, Austria (EK Nr AN2014-0312 343/4.6) and the ethics committee of “Land Vorarlberg” (EK-2-9/2020). Signed informed consent was obtained from all subjects involved in the study. Consent for publication: Not applicable. Animal research: Not applicable. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Efficient prediction of blastocyst formation from early-stage human embryos is imperative for improving the success rates of assisted reproductive technology (ART). Clinics transfer embryos at the blastocyst stage on Day-5 but Day-3 embryo transfer offers the advantage of a shorter culture duration, which reduces exposure to laboratory conditions, potentially enhancing embryonic development within a more conducive uterine environment and improving the likelihood of successful pregnancies. In this paper, we present a novel ResNet-GRU deep-learning model to predict blastocyst formation at 72 HPI. The model considers the time-lapse images from the incubator from Day 0 to Day 3. The model predicts blastocyst formation with a validation accuracy of 93% from the cleavage stage. The sensitivity and specificity are 0.97 and 0.77 respectively. The deep learning model presented in this paper will assist the embryologist in identifying the best embryo to transfer at Day 3, leading to improved patient outcomes and pregnancy rates in ART., Competing Interests: Declarations Conflict of interest The authors declare that they have no conflict of interest., (© 2024. The Author(s).)

One of the first organizing processes during animal development is the assembly of embryonic cells into epithelia. Common features unite epithelialization across select bilaterians, however, we know less about the molecular and cellular mechanisms that drive epithelial emergence in early branching nonbilaterians. In sea anemones, epithelia emerge both during embryonic development and after cell aggregation of dissociated tissues. Although adhesion is required to keep cells together, it is not clear whether cell polarization plays a role as epithelia emerge from disordered aggregates. Here, we use the embryos of the sea anemone Nematostella vectensis to investigate the evolutionary origins of epithelial development. We demonstrate that lateral cell polarization is essential for epithelial organization in both embryos and aggregates. With disrupted lateral polarization, cell contact in the aggregate is not sufficient to trigger epithelialization and further tissue development. Specifically, knockdown of the conserved lateral polarity and tumor suppressor protein Lethal giant larvae (Lgl) disrupts epithelia in developing embryos and impairs the capacity of dissociated cells to epithelialize from aggregates. In contrast to other systems, cells in Nematostella lgl knockdown embryos do not undergo excessive proliferation. Cells with reduced Lgl levels lose their columnar shape and proper positioning of their mitotic spindles and basal bodies. Due to misoriented divisions and aberrant shapes, cells arrange nonuniformly without forming a monolayer. Together our data show that, in Nematostella, Lgl drives epithelialization in embryos and cell aggregates through its effect on cell shape and organelle localization., Competing Interests: Competing interests statement:The authors declare no competing interest.

Oocytes spontaneously resume meiosis following their liberation from follicles, preventing full competence acquisition. Biphasic IVM (CAPA-IVM) maintains oocytes in meiotic arrest to improve developmental competence, and it specially affects poorly developed oocytes. We assessed the effect of CAPA-IVM on oocytes from small (<3mm) and large (>3mm) follicles of prepubertal goats. Oocytes were cultured for 6h in pre-IVM with C-type natriuretic peptide (CNP) and estradiol as meiotic inhibitors, and germinal vesicle (GV) rate and chromatin configuration were assessed. Then, oocytes were cultured in conventional IVM (c-IVM) or CAPA-IVM (pre-IVM + c-IVM) and EGF receptor (EGFR) protein expression, intra-oocyte ROS and blastocyst development were assessed. GV rate was higher in CNP groups than control (69% vs 28%, and 67% vs 31%, small and large follicles, respectively), but GV chromatin configuration was similar. In large follicles, EGFR expression was higher in oocytes and cumulus cells after CAPA-IVM, and ROS levels were lower. In small follicles these differences were not observed. c-IVM and CAPA-IVM produced similar blastocyst rates in small (3.7% vs 2.6%, respectively) and large follicles (8.3% vs 2.5%). Overall, CAPA-IVM enhanced EGFR expression for EGF peptide signalling and antioxidant capacity in oocytes from large follicles but oocytes from small follicles were too immature to benefit from it.

The Arabian goby, Cryptocentroides arabicus (Gobiidae: Gobiinae), is a significant species in marine and brackish water ecosystems. However, limited information is available about the reproductive biology and the early life history of this species. This study aimed to understand the embryonic and pre-larval development of C. arabicus under controlled laboratory conditions using light microscopy. Ten fish pairs were placed in five 70-L glass aquaria, and environmental parameters were carefully monitored. After 4 months, spawning began, with each event yielding between 700 and 1500 eggs, totaling 10 spawning events. The elliptical-shaped eggs, measuring 3.9 ± 0.1 mm in length and 1.6 mm in maximum width, hatched after 7 days and 4 h at a water temperature of 28°C. This process resulted in the identification of eight distinct larval developmental stages. Parental care by both the male and female was observed during the incubation period. Documenting the morphological changes during incubation contributes to a better understanding of the reproductive behaviour and early life history of C. arabicus. This is essential for the conservation management of marine and brackish water environments., (© 2024 Wiley‐VCH GmbH. Published by John Wiley & Sons Ltd.)

Border-associated macrophages (BAMs) play a pivotal role in maintaining brain homeostasis and responding to pathological conditions. Understanding their origins, characteristics, and roles in both healthy and diseased brains is crucial for advancing our knowledge of neuroinflammatory and neurodegenerative diseases. This review addresses the ontogeny, replenishment, microenvironmental regulation, and transcriptomic heterogeneity of BAMs, highlighting recent advancements in lineage tracing and fate-mapping studies. Furthermore, we examine the roles of BAMs in maintaining brain homeostasis, immune surveillance, and responses to injury and neurodegenerative diseases. Further research is crucial to clarify the dynamic interplay between BAMs and the brain's microenvironment in health and disease. This effort will not only resolve existing controversies but also reveal new therapeutic targets for neuroinflammatory and neurodegenerative disorders, pushing the boundaries of neuroscience., (© 2024 The Author(s). CNS Neuroscience & Therapeutics published by John Wiley & Sons Ltd.)

Muscle loading is known to influence skeletal morphology. Therefore, modification of the biomechanical environment is expected to cause coordinated morphological changes to the bony and cartilaginous tissues. Understanding how this musculoskeletal coordination contributes to morphological variation has relevance to health sciences, developmental biology, and evolutionary biology. To investigate how muscle loading influences skeletal morphology, we replicate a classic in ovo embryology experiment in the domestic chick (Gallus gallus domesticus) while harnessing modern methodologies that allow us to quantify skeletal anatomy more precisely and in situ. We induced rigid muscle paralysis in developing chicks mid-incubation, then compared the morphology of the cranium and mandible between immobilized and untreated embryos using microcomputed tomography and landmark-based geometric morphometric methods. Like earlier studies, we found predictable differences in the size and shape of the cranium and mandible in paralyzed chicks. These differences were concentrated in areas known to experience high strains during feeding, including the jaw joint and jaw muscle attachment sites. These results highlight specific areas of the skull that appear to be mechanosensitive and suggest muscles that could produce the biomechanical stimuli necessary for normal hatchling morphology. Interestingly, these same areas correspond to areas that show the greatest disparity and fastest evolutionary rates across the avian diversity, which suggests that the musculoskeletal integration observed during development extends to macroevolutionary scales. Thus, selection and evolutionary changes to muscle physiology and architecture could generate large and predictable changes to skull morphology. Building upon previous work, the adoption of modern imaging and morphometric techniques allows richer characterization of musculoskeletal integration that empowers researchers to understand how tissue-to-tissue interactions contribute to overall phenotypic variation., (© 2024 Wiley Periodicals LLC.)

Study Question: What are the implications of the presence cytoplasmic strings (Cyt-S) and their quantity and dynamics for the pre-implantation development of human blastocysts?, Summary Answer: Cyt-S are common in human embryos and are associated with faster blastocyst development, larger expansion, and better morphological quality., What Is Known Already: Cyt-S are dynamic cellular projections connecting inner cell mass and trophectoderm (TE) cells, that can be observed during blastocyst expansion. Their prevalence in human embryos has been estimated to be between 44% and 93%. Data relevant to their clinical implications and role in development are lacking, limited, or controversial., Study Design, Size, Duration: Retrospective study conducted at a single IVF center between May 2013 and November 2014 and involving 124 pre-implantation genetic testing for aneuploidy cycles in a time-lapse incubator with ≥1 blastocyst biopsied and vitrified (N = 370 embryos assessed). These cycles resulted in 87 vitrified-warmed single-euploid blastocyst transfers., Participants/materials, Setting, Methods: ICSI, continuous blastocyst culture (Days 5-7), TE biopsy of fully expanded blastocysts without Day 3 zona pellucida drilling, qPCR to assess uniform full-chromosome aneuploidies, and vitrification were all performed. Only vitrified-warmed euploid single-embryo-transfers were conducted. Blastocyst morphological quality was defined according to Gardner's criteria. The AI-based software CHLOE™ (Fairtility) automatically registered timings from time of starting blastulation (tSB) to biopsy (t-biopsy, i.e. blastocyst full-expansion) as hours-post-insemination (hpi), embryo area (including zona pellucida in µm2), and spontaneous blastocyst collapses. One senior embryologist manually annotated Cyt-S presence, quantity, timings, and type (thick cell-to-cell connections and/or threads). All significant associations were confirmed through regression analyses. All couples', cycles', and embryos' main features were also tested for associations with Cyt-S presence, quantity, and dynamics., Main Results and the Role of Chance: About 94.3% of the patients (N = 117/124) had ≥1 embryo with Cyt-S. Out of a total of 370 blastocysts, 55 degenerated between blastulation and full-expansion (N = 55/370, 14.9%). The degeneration rate among embryos with ≥1 Cyt-S was 10.8% (N = 33/304), significantly lower than that of embryos without Cyt-S (33.3%, N = 22/66, P < 0.01). Of the remaining 315 viable blastocysts analyzed, 86% (N = 271/315; P < 0.01) had ≥1 Cyt-S, on average 3.5 ± 2.1 per embryo ranging 1-13. The first Cyt-S per viable embryo appeared at 115.3 ± 12.5 hpi (85.7-157.7), corresponding to 10.5 ± 5.8 h (0.5-31) after tSB. Overall, we analyzed 937 Cyt-S showing a mean duration of 3.8 ± 2.7 h (0.3-20.9). Cyt-S were mostly threads (N = 508/937, 54.2%) or thick cell-to-cell connections becoming threads (N = 382/937, 40.8%) than thick bridges (N = 47/937, 5.0%). The presence and quantity of Cyt-S were significantly associated with developmentally faster (on average 6-12 h faster) and more expanded (on average 2700 µm2-larger blastocyst's area at t-biopsy) embryos. Also, the presence and duration of Cyt-S were associated with better morphology. Lastly, while euploidy rates were comparable between blastocysts with and without Cyt-S, all euploid blastocysts transferred from the latter group failed to implant (N = 10)., Limitations, Reasons for Caution: Cyt-S presence and dynamics were assessed manually on seven focal planes from video frames recorded every 15 min. The patients included were mostly of advanced maternal age. Only associations could be reported, but no causations/consequences. Lastly, larger datasets are required to better assess Cyt-S associations with clinical outcomes., Wider Implications of the Findings: Cyt-S are common during human blastocyst expansion, suggesting their physiological implication in this process. Their presence, quantity and dynamics mirror embryo viability, and morphological quality, yet their role is still unknown. Future basic science studies are encouraged to finally describe Cyt-S molecular nature and biophysical properties, and Artificial Intelligence tools should aid these studies by incorporating Cyt-S assessment., Study Funding/competing Interest(s): None., Trial Registration Number: N/A., (© The Author(s) 2024. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Increasing evidence suggests that environmental exposures can modify epigenetic marks in the germline, leading to the transmission of abnormal post-fertilization sperm epigenetic indicators and affecting embryonic development. Given the pivotal role of sperm cells in determining embryo quality, there is growing interest in understanding the potential effects of sperm sex sorting on embryo quality. This study aimed to investigate the impact of bovine sperm sexing on in vitro embryo production (IVP) and to associate molecular aspects of embryos analysis. Frozen semen samples from five Nellore bulls were used, with each bull contributing unsexed sperm (conventional semen - CV treatment) and female and male sexed sperm pooled after thawing (SX treatment). First, semen quality was assessed, including motility, morphology, acrosome integrity, and chromatin integrity to denaturation. Then, IVP was carried out, focusing on embryonic production and developmental kinetics. In the third experiment, embryo quality was evaluated by examining the gene expression of key markers (OCT4, NANOG, DNMT3A, TET1, and Fematrin-1) and the methylation pattern of the Satellite-1 and α-Satellite genes in blastocysts. Differences between CV and SX semen were only observed in motility, which was lower in SX compared with CV (P < 0.05). Although cleavage was similar, the SX groups showed lower blastocyst production than CV (P < 0.05). Of the genes evaluated, only NANOG showed high expression in the CV blastocysts compared with the SX blastocysts, but the methylation pattern revealed no differences. In conclusion, sex sorting markedly affects sperm motility and in vitro embryo production but showed no significant impact on embryo quality., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Research Question: What is the effect of embryo morphology score on 14-day β-HCG levels and 14-18-day β-HCG doubling values, and do they have differences in day-3 embryo or day-5 blastocyst transfers?, Design: Retrospective analysis of 4434 fresh cycles of single embryo transfers (SET) with β-HCG ≥15 mIU/ml on day 14 after transfer via IVF and ICSI. The correlation between embryo morphology score and 14-day β-HCG was examined. Doubling of 14-18 day β-HCG was analysed in 2628 cycles to determine correlations with embryo morphology score., Results: In day-3 SET, number of embryonic cells was positively correlated with 14-day post-transfer β-HCG values (R = 0.076; P = 0.013). No significant correlation was observed between the grade of the transferred embryos and the 14-18-day serum β-HCG doubling values. In day-5 single blastocyst transfers, the degree of blastocyst expansion, trophoblast cell and inner cell mass (ICM) grades demonstrated a significant positive correlation with 14-day post-transfer β-HCG (P < 0.001, P = 0.014, P = 0.003). Degree of blastocyst expansion was significantly correlated with 14-18-day β-HCG doubling values (R = -0.051, P = 0.027). Grades of the ICM and trophoblast cells showed no significant correlation with 14-18-day β-HCG doubling values., Conclusion: In fresh SET, embryo morphology score influences 14-day β-HCG values in day-3 embryos and day-5 blastocyst transfers. Embryo morphology score in day-3 SET does not affect 14-18-day β-HCG doubling values. Degree of blastocyst expansion significantly affects 14-18-day β-HCG doubling values in day-5 blastocyst transfers., (Copyright © 2024 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)

Objective: Multinucleated blastomeres at the two-cell stage (2MNB) represent a frequently observed nuclear abnormality in early human embryos. This abnormality has been reported to significantly impact on the embryo's developmental potential to reach the blastocyst stage. However, our understanding of the embryo's developmental potential and the morphokinetics of 2MNB remains limited. This study investigates the influence of 2MNB and its subtypes on the blastocyst formation., Study Design: A non-interventional retrospective study was performed in the Reproductive Medical Center of the First Affiliated Hospital of Hainan Medical University, using a time-lapse incubator. The study involved the evaluation of 4416 embryos, including 628 multinucleated embryos, from 1521 intracytoplasmic sperm injection (ICSI) cycles conducted between October 2019 and October 2021. The morphokinetic characteristics of multinucleated embryos were analyzed., Results: The results show multinucleation was the most common abnormal mitotic event during embryo development (14.22 %) in 4416 embryos. A control group of 3210 developmentally normal embryos was used in the study. The multinucleated blastomeres caused a lower blastocyst rate (52.48 % VS 64.02 %) compared to the control group. Whereas, 2MNBcause a higher blastocyst rate thanthemat the 4-cell stage (4MNB) (58.89 % VS 43.64 %). 2MNB can be further be further divided into 2MNB 1/2cell and 2MNB 2/2cell based on one multinucleated blastomere or two multinucleated blastomere appeared. Time to pronuclei fading (tPNf) is significantly longer in 2MNB 2/2cell compared to 2MNB 1/2cell . Furthermore, the 2MNB 1/2cell embryos were divided into four subgroups (Bi-: two nuclei with almost the same size, Micro-: two nuclei with varying sizes, Poly-: more than two nuclei with almost the same size, and Cluster-: more than two nuclei with varying sizes) based on the number of nuclei and relative size. The results show that the Bi- and Micro- groups had a significantly increased blastocyst rate. The Cluster-, and Poly- groups showed significantly delayed embryonic development compared to normal controls. Bi-group has significant delays at t3, t5, and t8 and the Micro-group had a significant delay only at t8., Conclusion: 2MNB cause higher blastocyst rate than them at 4MNB. 2MNB 1/2cell shows shorter tPNf compared to 2MNB 2/2cell . Moreover, the Micro-, Bi- groups had a significantly increased blastocyst rate and different kinetic parameters compared to Cluster-, Poly-groups, suggesting that it is necessary to distinguish the nucleus status within 2MNB to increase the blastocyst rate. When selecting embryos for transformation from the 2MNB 1/2cell , Micro- is the best choice., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Although the knee joint (KNJ) and temporomandibular joint (TMJ) all belong to the synovial joint, there are many differences in developmental origin, joint structure and articular cartilage type. Studies of joint development in embryos have been performed, mainly using poultry and rodents. However, KNJ and TMJ in poultry and rodents differ from those in humans in several ways. Very little work has been done on the embryonic development of KNJ and TMJ in large mammals. Several studies have shown that pigs are ideal animals for embryonic development research. Embryonic day 30 (E30), E35, E45, E55, E75, E90, Postnatal day 0 (P0) and Postnatal day 30 (P30) embryos/fetuses from the pigs were used for this study. The results showed that KNJ develops earlier than TMJ. Only one mesenchymal condensate of KNJ is formed on E30, while two mesenchymal condensates of TMJ are present on E35. All structures of KNJ and TMJ were formed on E45. The growth plate of KNJ begins to develop on E45 and becomes more pronounced from E55 to P30. From E75 to E90, more and more vascular-rich cartilage canals form in the cartilage regions of both joints. The cartilaginous canal of the TMJ divides the condyle into sections along the longitudinal axis of the condyle. This arrangement of cartilaginous canal was not found in the KNJ. The chondrification of KNJ precedes that of TMJ. Ossification of the knee condyle occurs gradually from the middle to the periphery, while that of the TMJ occurs gradually from the base of the mandibular condyle. In the KNJ, the ossification of the articular condyle is evident from P0 to P30, and the growth plate is completely formed on P30. In the TMJ, the cartilage layer of condyle becomes thinner from P0 to P30. There is no growth plate formation in TMJ during its entire development. There is no growth plate formation in the TMJ throughout its development. The condyle may be the developmental center of the TMJ. The chondrocytes and hypertrophic chondrocytes of the growth plate are densely arranged. The condylar chondrocytes of TMJ are scattered, while the hypertrophic chondrocytes are arranged. Embryonic development of KNJ and TMJ in pigs is an important bridge for translating the results of rodent studies to medical applications.

Calcium ions (Ca 2+ ) regulate cell proliferation and differentiation and participate in various physiological activities of cells. The calcium transfer protein inositol 1,4,5-triphosphate receptor (IP 3 R), located between the endoplasmic reticulum (ER) and mitochondria, plays an important role in regulating Ca 2+ levels. However, the mechanism by which IP 3 R1 affects porcine meiotic progression and embryonic development remains unclear. We established a model in porcine oocytes using siRNA-mediated knockdown of IP 3 R1 to investigate the effects of IP 3 R1 on porcine oocyte meiotic progression and embryonic development. The results indicated that a decrease in IP 3 R1 expression significantly enhanced the interaction between the ER and mitochondria. Additionally, the interaction between the ER and the mitochondrial Ca 2+ ([Ca 2+ ] m ) transport network protein IP 3 R1-GRP75-VDAC1 was disrupted. The results of the Duolink II in situ proximity ligation assay (PLA) revealed a weakened pairwise interaction between IP 3 R1-GRP75 and VDAC1 and a significantly increased interaction between GRP75 and VDAC1 after IP 3 R1 interference, resulting in the accumulation of large amounts of [Ca 2+ ] m . These changes led to mitochondrial oxidative stress, increased the levels of reactive oxygen species (ROS) and reduced ATP production, which hindered the maturation and late development of porcine oocytes and induced apoptosis. Nevertheless, after treat with [Ca 2+ ] m chelating agent ruthenium red (RR) or ROS scavenger N-acetylcysteine (NAC), the oocytes developmental abnormalities, oxidative stress and apoptosis caused by Ca 2+ overload were improved. In conclusion, our results indicated IP 3 R1 is required for meiotic progression and embryonic development by regulating mitochondrial calcium and oxidative damage., (Copyright © 2024 Elsevier Inc. All rights reserved.)

There is an increased interest in the evolution and development of newts from the genus Triturus because: (1) morphological differentiation among the nine constituent species largely corresponds to different ecological preferences, (2) hybridization between different species pairs has various evolutionary outcomes in terms of life history traits and morphology, and (3) the genus expresses a balanced lethal system that causes arrested growth and death of half of the embryos. These features provide natural experimental settings for molecular, morphological, and life-history studies. Therefore, we produce a staging table for the Balkan crested newt (T. ivanbureschi). We provide detailed descriptions of 34 embryonic stages based on easily observable and interpretable external morphological characters, to ensure reproducibility. Compared with previous staging tables for Triturus, we include a vastly increased sample size and provide high-resolution photographs in lateral, ventral, and dorsal view, complemented by videos of specific developmental periods, and accompanied by detailed explanations on how to delineate the specific stages. Our staging table will serve as a baseline in comparative studies on Triturus newts: an emerging model system in evolutionary and developmental studies., (© 2024 The Author(s). Journal of Experimental Zoology Part B: Molecular and Developmental Evolution published by Wiley Periodicals LLC.)

Objectives: Despite some existing studies on the safety of high static magnetic fields (SMFs), the effects of ultra-high SMFs above 20.0 T for embryonic development in early pregnancy are absent. The objective of this study is to evaluate the influence of 16.8-22.0 T SMF on the development of zebrafish embryos, which will provide important information for the future application of ultra-high field magnetic resonance imaging (MRI)., Methods: Two-hour exposure to homogenous (0 T/m) 22.0 T SMF, or 16.8 T SMFs with 123.25 T/m spatial gradient of opposite magnetic force directions was examined in the embryonic development of 200 zebrafish. Their body length, heart rate, spontaneous tail-wagging movement, hatching and survival rate, photomotor response, and visual motor response (VMR) were analyzed., Results: Our results show that these ultra-high SMFs did not significantly affect the general development of zebrafish embryos, such as the body length or spontaneous tail-wagging movement. However, the hatching rate was reduced by the gradient SMFs (p < 0.05), but not the homogenous 22.0 T SMF. Moreover, although the zebrafish larva activities were differentially affected by these ultra-high SMFs (p < 0.05), the expression of several visual and neurodevelopmental genes (p < 0.05) was generally downregulated in the eyeball., Conclusions: Our findings suggest that exposure to ultra-high SMFs, especially the gradient SMFs, may have adverse effects on embryonic development, which should cause some attention to the future application of ultra-high field MRIs., Clinical Relevance Statement: As technology advances, it is conceivable that very strong magnetic fields may be adapted for use in medical imaging. Possible dangers associated with these higher Tesla fields need to be considered and evaluated prior to human use., Key Points: Ultra-High static magnetic field may affect early embryonic development. High strength gradient static magnetic field exposure impacted zebrafish embryonic development. The application of very strong magnetic fields for MR technologies needs to be carefully evaluated., (© 2024. The Author(s), under exclusive licence to European Society of Radiology.)

Cell cycle regulation is crucial to assure expansion of a cell population, while preserving genome integrity. This notion is especially relevant to fertilization and early embryo development, a time when the cell cycle transforms from meiotic into mitotic cycles. Zygote-to-embryo transition is acutely error-prone, causing major developmental perturbations, including cleavage delays, tri- and multi-chotomous cleavages, and cell fragmentation. Another such alteration is bi- and multinucleation, consisting of the simultaneous formation of two or more nuclei at interphase. Indeed, multinucleation affects a large proportion of early human embryos, typically at the two-cell stage. Mechanistically, several factors, including spindle dysfunction, failed cleavage, and cell fusion, may generate this cell anomaly. In assisted reproduction treatment, multinucleation is associated with reduced developmental rates and lower implantation rates in Days 2-3 embryo transfers. However, many multinucleated embryos can develop to the blastocyst stage. In blastocyst transfers, the current evidence does not suggest a major impact of a previous history of multinucleation on the odds of euploidy or successful treatment outcomes. Human embryo multinucleation remains a not-fully-understood but developmentally relevant and intriguing phenomenon which requires further research of its generative mechanisms and clinical implications., (© The Author(s) 2024. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

During embryonic development, cells shape our body, which is mostly made up of water. It is often forgotten that some of this water is found in intercellular fluid, which, for example, immerses the cells of developing embryos. Intercellular fluid contributes to the properties of tissues and influences cell behaviour, thereby participating in tissue morphogenesis. While our understanding of the role of cells in shaping tissues advances, the exploration of the contribution of intercellular fluid dynamics is just beginning. In this review, we delve into the intricate mechanisms employed by cells to control fluid movements both across and within sealed tissue compartments. These mechanisms encompass sealing by tight junctions and controlled leakage, osmotic pumping, hydraulic fracturing of cell adhesion, cell and tissue contractions, as well as beating cilia. We illustrate key concepts by drawing extensively from the early mouse embryo, which successively forms multiple lumens that play essential roles in its development. Finally, we detail experimental approaches and emerging techniques that allow for the quantitative characterization and the manipulation of intercellular fluids in vivo, as well as theoretical frameworks that are crucial for comprehending their dynamics., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Background: Melatonin, a hormone found in various bodily fluids and cells, is known for its potent antioxidative, anti-apoptotic, and endocrine regulatory properties. This study aimed to analyze melatonin levels in patients with diminished ovarian reserve (DOR) and its impact on embryo quality., Methods: We enrolled 85 women who were undergoing in vitro fertilization or intracytoplasmic sperm injection procedures, including normal ovarian reserve (NOR, n = 27), pathological DOR (DOR-Path, n = 25), and physiological DOR (DOR-Phy, n = 33). Melatonin levels in patient serum and follicular fluid were assessed using ELISA, and correlations between melatonin levels and indicators of embryo quality were examined., Results: Our findings indicate that melatonin levels in the follicular fluid and basal serum of the DOR-Path and DOR-Phy groups were lower compared to the NOR group (P < 0.05). However, no significant differences in melatonin levels were found between the DOR-Path and DOR-Phy groups (P > 0.05). Additionally, the concentration of melatonin in the follicular fluid of the NOR group was significantly higher than in their serum (P < 0.001). Lastly, a significant correlation was discovered between melatonin levels in serum and follicular fluid and parameters of ovarian reserve and embryonic development (P < 0.05)., Conclusions: Melatonin levels in DOR patients may impact embryo quality, offering insights into potential DOR pathogenesis and opportunities to enhance treatment outcomes in these patients., (© 2024. The Author(s).)

The subcortical maternal complex (SCMC) is essential for safeguarding female fertility in mammals. Assembled in oocytes, the SCMC maintains the cleavage of early embryos, but the underlying mechanism remains unclear. Here, we report that 14-3-3, a multifunctional protein, is a component of the SCMC. By resolving the structure of the 14-3-3-containing SCMC, we discover that phosphorylation of TLE6 contributes to the recruitment of 14-3-3. Mechanistically, during maternal-to-embryo transition, the SCMC stabilizes 14-3-3 protein and contributes to the proper control of CDC25B, thus ensuring the activation of the maturation-promoting factor and mitotic entry in mouse zygotes. Notably, the SCMC establishes a conserved molecular link with 14-3-3 and CDC25B in human oocytes/embryos. This study discloses the molecular mechanism through which the SCMC regulates the cell cycle in early embryos and elucidates the function of the SCMC in mammalian early embryogenesis., (© 2024. The Author(s).)

The emergence of the earliest cell lineages in mammalian embryos is a complex process that utilizes an extensive network of chromatin regulators, transcription factors, cell polarity regulators, and cellular signaling pathways. These factors and pathways operate over a protracted period of time as embryos cleave, undergo compaction, and form blastocysts. The first cell fate specification event separates the pluripotent inner cell mass from the trophectoderm lineage. The second event separates pluripotent epiblast from hypoblast. This review summarizes over 50 years of study of these early lineage forming events, addressing the complexity of the network of interacting molecules, cellular functions and pathways that drive them, interspecies differences, and aspects of these mechanisms that likely underlie their high susceptibility to disruption by numerous environmental factors that can compromise embryo viability, such as maternal health and diet, environmental toxins, and other stressors., (© 2024 The Author(s). Molecular Reproduction and Development published by Wiley Periodicals LLC.)

Chicken embryos have many advantages in the study of amniote embryonic development. In particular, culture techniques developed for early-stage embryos have promoted the advancement of modern developmental studies using chicken embryos. However, the standard technique involves placing chicken embryos in the ventral-upward (ventral-up) orientation, limiting manipulation of the epiblast at the dorsal surface, which is the primary source of ectodermal and mesodermal tissues. To circumvent this limitation, we developed chicken embryo cultures in the dorsal-up orientation and exploited this technique to address diverse issues. In this article, we first review the history of chicken embryo culture techniques to evaluate the advantages and limitations of the current standard technique. Then, the dorsal-up technique is discussed. These technological discussions are followed by three different examples of experimental analyses using dorsal-up cultures to illustrate their advantages: (1) EdU labeling of epiblast cells to assess potential variation in the cell proliferation rate; (2) migration behaviors of N1 enhancer-active epiblast cells revealed by tracking cells with focal fluorescent dye labeling in dorsal-up embryo culture; and (3) neural crest development of mouse neural stem cells in chicken embryos., (© 2024 The Author(s). Development, Growth & Differentiation published by John Wiley & Sons Australia, Ltd on behalf of Japanese Society of Developmental Biologists.)

Our understanding of the processes of human development that occur during and just after implantation is still incomplete. The anatomical studies by Erich Blechschmidt (1904-1992) at the University of Göttingen demonstrate the uniqueness and beauty of the early stages of individual human development or ontogeny. The interpretations of human embryology by Blechschmidt offer a simple unifying hypothesis: metabolic and biomechanical events are repeated, and this can be described as an ontogenetic recapitulation. This commentary provides a rationale for using some older terms and introducing new ones in the description of early human development. The product of conception is a conceptus; the outer part of the conceptus is the ectoblast and everything inside is the endoblast; the endocyst arises in the endoblast when the future amniotic fluid is forming. The human embryo arises from the innermost part of the endocyst. Terms such as morula, gastrula, and cyema, which are imported from zoology and ignore the role of the zona pellucida and constrained fluid compartments in the conceptus, can be avoided., (© 2024 The Author(s). Clinical Anatomy published by Wiley Periodicals LLC on behalf of American Association of Clinical Anatomists and British Association of Clinical Anatomists.)

During the fetal period, oxygenated blood from the placenta flows through the umbilical vein (UV), portal sinus, ductus venosus (DV), and inferior vena cava (IVC) to the heart. This venous route varies regionally in many aspects. Herein, we sought to characterize the venous route's morphological features and regional differences during embryonic and early-fetal periods. Twenty-nine specimens were selected for high-resolution digitized imaging; 18 embryos were chosen for histological analysis. The venous route showed a primitive, large, S-shaped curved morphology with regional narrowing and dilation at Carnegie stage (CS) 15. Regional differences in vessel-wall differentiation became apparent from approximately CS20. The vessel wall was poorly developed in most DV parts; local vessel-wall thickness at the inlet was first detected at CS20. The lumen of the venous route changed from a nonuniform shape to a relatively round and uniform morphology after CS21. During the early-fetal period, two large bends were observed around the passage of the umbilical ring and at the inlet of the liver. The length ratio of the extrahepatic UV to the total venous route increased. The sectional area gradually increased during embryonic development, whereas differences in sectional area between the DV, UV, and IVC became more pronounced in the early-fetal period. Furthermore, differences in the sectional area between the narrowest part of the DV and other hepatic veins and the transverse sinus became more pronounced. In summary, the present study described morphological, morphometric, and histological changes in the venous route throughout embryonic and early-fetal development, clarifying regional characteristics., (© 2024 American Association for Anatomy.)

Thermal performance curves (TPCs) provide a framework for understanding the effects of temperature on ectotherm performance and fitness. TPCs are often used to test hypotheses regarding local adaptation to temperature or to develop predictions for how organisms will respond to climate warming. However, for aquatic organisms such as fishes, most TPCs have been estimated for adult life stages, and little is known about the shape of TPCs or the potential for thermal adaptation at sensitive embryonic life stages. To examine how latitudinal gradients shape TPCs at early life stages in fishes, we used two populations of Fundulus heteroclitus that have been shown to exhibit latitudinal variation along the thermal cline as adults. We exposed embryos from both northern and southern populations and their reciprocal crosses to eight different temperatures (15°C, 18°C, 21°C, 24°C, 27°C, 30°C, 33°C, and 36°C) until hatch and examined the effects of developmental temperature on embryonic and larval traits (shape of TPCs, heart rate, and body size). We found that the pure southern embryos had a right-shifted TPC (higher thermal optimum (T opt ) for developmental rate, survival, and embryonic growth rate) whereas pure northern embryos had a vertically shifted TPC (higher maximum performance (P max ) for developmental rate). Differences across larval traits and cross-type were also found, such that northern crosses hatched faster and hatched at a smaller size compared to the pure southern population. Overall, these observed differences in embryonic and larval traits are consistent with patterns of both local adaptation and countergradient variation., (© 2024 The Author(s). Journal of Experimental Zoology Part A: Ecological and Integrative Physiology published by Wiley Periodicals LLC.)

Mitochondria are commonly recognized as the powerhouses of the cell, primarily responsible for energy production through oxidative phosphorylation. Alongside this vital function, they also play crucial roles in regulating calcium signaling, maintaining membrane potential, and modulating apoptosis. Their involvement in various cellular pathways becomes particularly evident during oogenesis and embryogenesis, where mitochondrial quantity, morphology, and distribution are tightly controlled. The efficiency of the mitochondrial network is maintained through multiple quality control mechanisms that are essential for reproductive success. These include mitochondrial unfolded protein response, mitochondrial dynamics, and mitophagy. Not surprisingly, mitochondrial dysfunction has been implicated in infertility and ovarian aging, prompting investigation into mitochondria as diagnostic and therapeutic targets in assisted reproduction. To date, mitochondrial DNA copy number in oocytes, cumulus cells, and trophectoderm biopsies, and fluorescent lifetime imaging microscopy-based assessment of NADH and flavin adenine dinucleotide content have been explored as potential predictors of embryo competence, yielding limited success. Despite challenges in the clinical application of mitochondrial diagnostic strategies, these enigmatic organelles have a significant impact on reproduction, and their potential role as diagnostic targets in assisted reproduction is likely to remain an active area of investigation in the foreseeable future., (© The Author(s) 2024. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Physical parameters such as tissue interplay forces, luminal pressure, fluid flow, temperature, and electric fields are crucial regulators of embryonic morphogenesis. While significant attention has been given to cellular and molecular responses to these physical parameters, their roles in morphogenesis are not yet fully elucidated. This is largely due to a shortage of methods for spatiotemporal modulation and direct quantitative perturbation of physical parameters in embryos. Recent advancements addressing these challenges include microscopes equipped with devices to apply and adjust forces, direct perturbation of luminal pressure, and the application of micro-forces to targeted cells and cilia in vivo. These methods are critical for unveiling morphogenesis mechanisms, highlighting the importance of integrating molecular and physical approaches for a comprehensive understanding of morphogenesis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Formation of organs and specialized tissues in embryonic development requires migration of cells to specific targets. In some instances, such cells migrate as a robust cluster. We here explore a recent local approximation of non-local continuum models by Falcó et al. (SIAM J Appl Math 84:17-42, 2023). We apply their theoretical results by specifying biologically-based cell-cell interactions, showing how such cell communication results in an effective attraction-repulsion Morse potential. We then explore the clustering instability, the existence and size of the cluster, and its stability. For attractant-repellent chemotaxis, we derive an explicit condition on cell and chemical properties that guarantee the existence of robust clusters. We also extend their work by investigating the accuracy of the local approximation relative to the full non-local model., (© 2024. The Author(s), under exclusive licence to the Society for Mathematical Biology.)

Pre-implantation embryonic development occurs in the oviduct during the first few days of pregnancy. The presence of oviductal extracellular vesicles (oEVs, also called oviductosomes) is crucial for pre-implantation embryonic development in vivo as oEVs often contain molecular transmitters such as proteins. Therefore, evaluating oEV cargo during early pregnancy could provide insights into factors required for proper early embryonic development that are missing in the current in vitro embryo culture setting. In this study, we isolated oEVs from the oviductal fluid at estrus and different stages of early embryonic development. The 2306-3066 proteins in oEVs identified at the different time points revealed 58-60 common EV markers identified in exosome databases. Oviductal extracellular vesicle proteins from pregnant samples significantly differed from those in non-pregnant samples. In addition, superovulation changes the protein contents in oEVs compared to natural ovulation at estrus. Importantly, we have identified that embryo-protectant proteins such as high-mobility protein group B1 and serine (or cysteine) peptidase inhibitor were only enriched in the presence of embryos. We also visualized the physical interaction of EVs and the zona pellucida of 4- to 8-cell stage embryos using transmission electron microscopy as well as in vivo live imaging of epithelial cell-derived GFP-tagged CD9 mouse model. All protein data in this study are readily available to the scientific community in a searchable format at https://genes.winuthayanon.com/winuthayanon/oviduct&#95;ev&#95;proteins/. In conclusion, we identified oEVs proteins that could be tested to determine whether they can improve embryonic developmental outcomes in vivo and in vitro setting., (© 2024 Federation of American Societies for Experimental Biology.)

The efficiency of in vitro embryo production in mammals is influenced by variables associated with culture conditions during maturation, fertilization, and embryonic development. The embryos obtained often exhibit low quality due to suboptimal in vitro culture conditions compared to the in vivo environment. Co-culturing gametes and embryos with somatic cells has been developed to enhance in vitro culture conditions. This study aimed to assess the impact of coculturing in vitro-produced porcine embryos with porcine oviductal epithelial cells (POEC) on embryo development and quality. Firstly, a pure culture of POEC suitable for coculture systems was established. The epithelial origin of the cells was confirmed by the expression of E-cadherin and cytokeratin. The expression pattern of hormone receptors aligned with the diestrous oviduct, and POEC also secreted oviductal glycoprotein type 1 (OVGP-1). Secondly, POEC from passage 1 (POEC-1) were used to coculture with in vitro-produced porcine embryos. A successful coculture system was established without the addition of fetal bovine serum as a supplement. Coculturing POEC-1 in monolayers with in vitro-produced porcine embryos during the initial two days of culture enhanced the percentage of blastocysts and their hatching. Although the coculture did not alter the number of cells in the blastocysts or apoptosis assessed by TUNEL, it significantly reduced reactive oxygen species (ROS) levels in cleaved porcine embryos. This study represents the first report evaluating the quality of porcine embryos produced by IVF in coculture systems and assessing ROS levels in cleaved porcine embryos obtained by IVF., (Copyright © 2024 Elsevier Inc. All rights reserved.)

In pigs, the majority of embryonic mortality occurs when free-floating conceptuses (embryos/fetuses and associated placental membranes) elongate, and the uterine-placental interface undergoes folding and develops areolae. Both periods involve proliferation, migration, and changes in morphology of cells that require adenosine triphosphate (ATP). We hypothesize that insufficient ATP in conceptus and uterine tissues contributes to conceptus loss in pigs. Creatine is stored in cells as phosphocreatine for ATP regeneration through the creatine-creatine kinase- phosphocreatine pathway. However, the expression of components of this pathway in pigs has not been examined throughout gestation. Results of qPCR analyses indicated increases in AGAT, GAMT, CKM, CKB, and SLC6A8 mRNAs in elongating porcine conceptuses, and immunofluorescence microscopy localized guanidinoacetate N-methyltransferase, creatine kinase M, and creatine kinase B proteins to the trophectoderm of elongating conceptuses, to the columnar chorionic epithelial cells at the bottom of chorioallantoic troughs, and to endometrial luminal epithelium at the tops of the endometrial ridges of uterine-placental folds on Days 40, 60, and 90 of gestation. Guanidinoacetate N-methyltransferase protein is expressed in endometrial luminal epithelium at the uterine-placental interface, but immunostaining is more intense in luminal epithelium at the bottoms of the endometrial ridges. Results of this study indicate that key elements of the pathway for creatine metabolism are expressed in cells of the conceptus, placenta, and uterus for potential production of ATP during two timepoints in pregnancy with a high demand for energy; elongation of the conceptus for implantation and development of uterine-placental folding during placentation., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Following blastocyst hatching, ungulate embryos undergo a prolonged preimplantation period termed conceptus elongation. Conceptus elongation constitutes a highly susceptible period for embryonic loss, and the embryonic requirements during this process are largely unknown, but multiple lipid compounds have been identified in the fluid nourishing the elongating conceptuses. Peroxisome proliferator-activated receptors mediate the signaling actions of prostaglandins and other lipids, and, between them, PPARG has been pointed out to play a relevant role in conceptus elongation by a functional study that depleted PPARG in both uterus and conceptus. The objective of this study has been to determine if embryonic PPARG is required for bovine embryo development. To that aim, we have generated bovine PPARG knock-out embryos in vitro using two independent gene ablation strategies and assessed their developmental ability. In vitro development to Day 8 blastocyst was unaffected by PPARG ablation, as total, inner cell mass, and trophectoderm cell numbers were similar between wild-type and knock-out D8 embryos. In vitro post-hatching development to D12 was also comparable between different genotypes, as embryo diameter, epiblast cell number, embryonic disk formation, and hypoblast migration rates were unaffected by the ablation. The development of tubular stages equivalent to E14 was assessed in vivo, following a heterologous embryo transfer experiment, observing that the development of extra-embryonic membranes and of the embryonic disk was not altered by PPARG ablation. In conclusion, PPARG ablation did not impaired bovine embryo development up to tubular stages., (© The Author(s) 2024. Published by Oxford University Press behalf of Society for the Study of Reproduction.)

Thyroid hormone (TH) is essential for growth and development, yet its specific role during embryogenesis remains incompletely understood. This study investigates the impact of TH deficiency, induced by thiourea, a known inhibitor of thyroid peroxidase (TPO), on the development of domestic chicks. Thiourea was administered before thyroid gland formation, and its presence in treated embryos was confirmed through liquid chromatography-mass spectrometry. In silico docking revealed a strong interaction between thiourea and the CCP-like domain of TPO, which was corroborated by TPO activity assays showing reduced enzyme function. This reduction in enzyme activity led to lower embryonic TH levels and increased thyroid-stimulating hormone (TSH) secretion. Morphological analysis of newly hatched chicks revealed significant structural anomalies, particularly in lateral plate mesoderm-derived structures, including omphalocele, limb deformities, anophthalmia and craniofacial defects. Alcian blue and Alizarin red staining demonstrated reduced ossification in ribs and forelimbs, while histological analysis showed incomplete abdominal wall closure and abnormal vertebral column development. Haematological profiling of TH-deficient newly hatched chicks revealed significantly lower blood cell counts, highlighting TH's critical role in haematopoiesis. These findings emphasise the multifaceted role of TH in embryonic development, with potential implications for understanding congenital hypothyroidism and its developmental impacts, especially in regions with limited healthcare access., (© 2024 Wiley‐VCH GmbH. Published by John Wiley & Sons Ltd.)

Context Sires differ in their ability to produce viable blastocysts, yet our understanding of the cellular mechanisms regulated by the sire during early embryo development is limited. Aims The first aim was to characterise autophagy and reactive oxygen species (ROS) in embryos produced by high and low performing sires under normal and stress culture conditions. The second aim was to evaluate DNA damage and lipid peroxidation as mechanisms that may be impacted by increased cellular stress, specifically oxidative stress. Methods Embryos were produced using four high and four low performing sires based on their ability to produce embryos. Autophagy and ROS were measured throughout development. To evaluate oxidative stress response, autophagy, and ROS were measured in 2-6 cell embryos exposed to heat stress. To understand how cellular stress impacts development, DNA damage and lipid peroxidation were assessed. Key results Under normal conditions, embryos from low performing sires had increased ROS and autophagy. Under heat stress, embryos from low performing sires had increased ROS, yet those from high performing sires had increased autophagy. There was no difference in DNA damage or lipid peroxidation. Conclusions Results suggest that embryos from low performing sires may begin development under increased cellular stress, and autophagy potentially increases to mitigate the impacts of stress. Implications There is potential for improving embryonic competence through selection of sires with lower stress-related markers.

Early amniote development is highly self-organized, capable of adapting to interference through local and long-range cell-cell interactions. This process, called embryonic regulation 1 , has been well illustrated in experiments on avian embryos, in which subdividing the epiblast disk into different parts not only redirects cell fates to eventually form a complete and well-proportioned embryo at its original location, but also leads to the self-organization of additional, fully formed embryos 2,3 in the other separated parts. The cellular interactions underlying embryonic self-organization are widely believed to be mediated by molecular signals, yet the identity of such signals is unclear. Here, by analysing intact and mechanically perturbed quail embryos, we show that the mechanical forces that drive embryogenesis self-organize, with contractility locally self-activating and the ensuing tension acting as a long-range inhibitor. This mechanical feedback governs the persistent pattern of tissue flows that shape the embryo 4-6 and also steers the concomitant emergence of embryonic territories by modulating gene expression, ensuring the formation of a single embryo under normal conditions, yet allowing the emergence of multiple, well-proportioned embryos after perturbations. Thus, mechanical forces act at the core of embryonic self-organization, shaping both tissues and gene expression to robustly yet plastically canalize early development., (© 2024. The Author(s).)

This study investigates the influence of four culture media pre-equilibration methods on embryo development and clinical pregnancy outcomes. The methods are as follows: Method A involved covering media with fresh mineral oil in humid-type incubators for 24 h. Method B replicated Method A in dry-type incubators. Method C utilized pre-equilibrated (humidified) mineral oil to cover the media, also in humid-type incubators for 24 h. Method D followed the same process as Method C but in dry-type incubators. Subsequently, media from all groups were transferred to dry-type incubators for 72 h. Osmolality was measured at 24, 48, 72, and 96 h. For G1 PLUS, no significant differences were observed among groups at 24, 48, and 72 h. However, at 96 h, Groups B and D exhibited significantly higher osmolality than Groups A and C (A vs B, p = 0.043; A vs D: p = 0.046; B vs C, p = 0.043; C vs D, p = 0.046). No significant variations were found between Groups A and C or B and D. Similar results were obtained for G2 PLUS. A retrospective analysis of embryo development and clinical outcomes using Methods A revealed significant improvements in good blastocysts and available embryos compared with Method B for all (p = 0.005 and 0.004) and IVF cycles (p = 0.025 and 0.017). Method A also enhanced blastocyst formation in ICSI cycles (p = 0.017). However, clinical pregnancy outcomes did not significantly differ between Methods A and B. Pre-equilibrating culture media overnight in humid-type incubators, even when covered with fresh mineral oil, significantly mitigates osmolality rise and improves embryo development potential during embryo culture in dry-type incubators., Competing Interests: Declaration of Competing Interest The authors declare that there is no conflict of interest., (Copyright © 2024 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier B.V. All rights reserved.)

In the first days of life, cells of the mammalian embryo segregate into two distinct lineages, trophectoderm and inner cell mass. Unlike nonmammalian species, mammalian development does not proceed from predetermined factors in the oocyte. Rather, asymmetries arise de novo in the early embryo incorporating cues from cell position, contractility, polarity, and cell-cell contacts. Molecular heterogeneities, including transcripts and non-coding RNAs, have now been characterized as early as the 2-cell stage. However, it's debated whether these early heterogeneities bias cells toward one fate or the other or whether lineage identity arises stochastically at the 16-cell stage. This review summarizes what is known about early blastomere asymmetries and our understanding of lineage allocation in the context of historical models. Preimplantation development is reviewed coupled with what is known about changes in morphology, contractility, and transcription factor networks. The addition of single-cell atlases of human embryos has begun to reveal key differences between human and mouse, including the timing of events and core transcription factors. Furthermore, the recent generation of blastoid models will provide valuable tools to test and understand fate determinants. Lastly, new techniques are reviewed, which may better synthesize existing knowledge with emerging data sets and reconcile models with the regulative capacity unique to the mammalian embryo., (© The Author(s) 2024. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Autophagy is essential for oocyte maturation and preimplantation embryo development. ATG4C, a member of the ATG4 family, plays a crucial role in the autophagy process. The effect of ATG4C on the early embryonic development in pig has not been studied. In this study, the expression patterns of ATG4C were explored using qRT-PCR and immunofluorescence staining. Different concentrations of serum were added to in vitro maturation (IVM) medium to investigate its effects on oocyte maturation and embryonic development. Finally, the developmental potential of parthenogenetic embryos was detected by downregulating ATG4C in MII stage oocytes under 0 % serum condition. The results revealed that ATG4C was highly expressed in porcine oocytes matured in vitro and in parthenogenetic embryos. Compared with the 10 % serum group, the cumulus cell expansion, first polar body (PB1) extrusion rate, and subsequent developmental competence of embryos were reduced in the 0 % and 5 % serum groups. The mRNA levels of LC3, ATG5, BECLIN1, TFAM, PGC1α, and PINK1 were significantly increased (P < 0.05) in the 0 % serum group. ATG4C was significantly upregulated in the embryos at the 1-cell, 2-cell, 8-cell, and 16-cell stages in the 0 % serum group (P < 0.05). Compared with the negative control group, downregulation of ATG4C significantly decreased the 4-cell, 8-cell, and blastocyst rates (P < 0.05), and the expression of genes related to autophagy, mitochondria, and zygotic genome activation (ZGA) was significantly decreased (P < 0.05). The relative fluorescence intensity of LC3 and mitochondrial content in the ATG4C siRNA group was significantly reduced (P < 0.05). Collectively, the results indicate that ATG4C is highly expressed in porcine oocytes matured in vitro and in early embryos, and inhibition of ATG4C effects embryonic developmental competence by decreasing autophagy, mitochondrial content, and ZGA under serum-free condition., Competing Interests: Declaration of competing interest None of the authors have any conflict of interest to declare., (Copyright © 2024 Elsevier Inc. All rights reserved.)

As development varies greatly across the tree of life, it may seem difficult to suggest a model that proposes a single mechanism for understanding collective cell behaviors and the coordination of tissue formation. Here we propose a mechanism called differentiation waves, which unify many disparate results involving developmental systems from across the tree of life. We demonstrate how a relatively simple model of differentiation proceeds not from function-related molecular mechanisms, but from so-called differentiation waves. A phenotypic model of differentiation waves is introduced, and its relation to molecular mechanisms is proposed. These waves contribute to a differentiation tree, which is an alternate way of viewing cell lineage and local action of the molecular factors. We construct a model of differentiation wave-related molecular mechanisms (genome, epigenome, and proteome) based on bioinformatic data from the nematode Caenorhabditis elegans. To validate this approach across different modes of development, we evaluate protein expression across different types of development by comparing Caenorhabditis elegans with several model organisms: fruit flies (Drosophila melanogaster), yeast (Saccharomyces cerevisiae), and mouse (Mus musculus). Inspired by gene regulatory networks, two Models of Interactive Contributions (fully-connected MICs and ordered MICs) are used to suggest potential genomic contributions to differentiation wave-related proteins. This, in turn, provides a framework for understanding differentiation and development., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

This review describes in detail the morphological, cytoskeletal and gene expression events leading to the gene regulatory network bifurcation point of trophoblast and inner cell mass cells in a variety of mammalian preimplantation embryos. The interrelated processes of compaction and polarity establishment are discussed in terms of how they affect YAP/WWTR activity and the location and fate of cells. Comparisons between mouse, human, cattle, pig and rabbit embryos suggest a conserved role for YAP/WWTR signalling in trophoblast induction in eutherian animals though the mechanisms for, and timing of, YAP/WWTR activation differs among species. Downstream targets show further differences, with the trophoblast marker GATA3 being a direct target in all examined mammals, while CDX2-positive and SOX2-negative regulation varies., (Copyright © 2024 The Author. Published by Elsevier Inc. All rights reserved.)

Low hatchability has been a persistent challenge in the goose industry. Establishing standard atlases and comprehending embryonic development patterns are essential to improving the hatching rates of goose eggs. However, comprehensive descriptions of normal atlases, embryonic development, and energy requirements in geese are lacking. In this study, a total of 120 fertile eggs from well-known large Shitou goose were incubated using 12 nesting purebred female geese. During hatching, both the temperature of the eggshells and the weight of eggs were recorded, and daily photographs of the embryos were captured to monitor their development closely. After hatching, counted the number of pores per unit area of eggshells by choosing eggs from without sperm, dead embryos, and normally hatched. Furthermore, 150 Shitou goose eggs were hatched by automatic incubator, with adjustments made based on observed normal developmental stages that incubated by female geese. The eggs were carefully opened to meticulously document embryonic morphology and create a detailed development map. Measurements were taken of the eye diameter, length of the lower beak, tarsometatarsus bone, and embryo length. Subsequently, an analysis was conducted to assess the calcium, phosphorus, crude protein, and crude fat content to study the energy requirements for embryo development. characteristics on the 7th, 15th, 23rd and 28th days of Shitou goose hatching corresponded to the 5th, 10th, 17th and 19th days of chicken egg incubation, respectively. These days were distinguished individually by "visible embryo's eye", "closure", "sealing the door", and "flashing hair". Besides, the hatch rate of the incubator reached 86.67%, and the cumulative water loss rate increased with embryo age. Notably, normally developing embryos displayed a significantly higher number of pores on the eggshell surface compared to dead embryos (P < 0.05). Additionally, embryonic body length, eyeball diameter, and lower beak length exhibited continuous growth until day 19 of incubation, while tarsometatarsus length increased steadily from days 12 to 31. Liver size measurement began on the 10th day of incubation, while both leg and chest muscles showed continuous growth from the 12th day. For energy demand, the embryo primarily relied on protein sourced from the egg yolk within the first 10 days of development. Afterward, the egg yolk provided both protein and fat for embryonic growth. In summary, this study has generated a comprehensive developmental map for Shitou goose embryos, offering valuable insights into their growth and morphological changes throughout the incubation period. This map can serve as a reference for optimizing machine incubation techniques to enhance goose egg hatching rates and provide fresh perspectives on the development of geese., Competing Interests: DISCLOSURES The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Regulatory cell death is an important way to eliminate the DNA damage that accompanies the rapid proliferation of neural stem cells during cortical development, including pyroptosis, apoptosis, and so on. Here, the study reports that the absence of GSDMD-mediated pyroptosis results in defective DNA damage sensor pathways accompanied by aberrant neurogenesis and autism-like behaviors in adult mice. Furthermore, GSDMD is involved in organizing the mitochondrial electron transport chain by regulating the AMPK/PGC-1α pathway to target Aifm3. This process promotes a switch from oxidative phosphorylation to glycolysis. The perturbation of metabolic homeostasis in neural progenitor cells increases lactate production which acts as a signaling molecule to regulate the p38MAPK pathway. And activates NF-𝜿B transcription to disrupt cortex development. This abnormal proliferation of neural progenitor cells can be rescued by inhibiting glycolysis and lactate production. Taken together, the study proposes a metabolic axis regulated by GSDMD that links pyroptosis with metabolic reprogramming. It provides a flexible perspective for the treatment of neurological disorders caused by genotoxic stress and neurodevelopmental disorders such as autism., (© 2024 The Author(s). Advanced Science published by Wiley‐VCH GmbH.)

ICSI is one of the most commonly used techniques to treat infertility. The sperm selection for the procedure is done 'randomly' by the embryologist according to the motility and morphology parameters which is known not to reflect the potential of a sperm for fertilization, pregnancy and a healthy childbearing. Since the apoptosis rate is higher in sperm cells of infertile patients, it is more likely to choose an apoptotic sperm by the 'random selection method'. We recently introduced a novel sperm selection technique namely 'Annexin-V coated polystrene bead technique'(APB-Tech), for the selection of non-apoptotic sperm cells. The principal of the technique is based on the binding affinity of an apoptotic sperm to 'Annexin-V covered beads' enabling to distinguish a viable and a healthy sperm by light microscopy. The aim of the present study was to observe the effects of this technique on ICSI outcomes in mice. Sibling-oocyte trial was conducted and the outcome measures were compared with the results of traditional sperm selection method. Embryo and blastocyst qualities and blastocyst development rates were significantly increased in APB-Tech group, while the other parameters were not affected. Promising results obtained from the technique reflect its promising potential as a new and powerful tool for sperm selection and thus infertility techniques., (© 2024. The Author(s).)

In mammals, early embryogenesis relies heavily on the regulation of maternal transcripts including protein-coding and non-coding RNAs stored in oocytes. In this study, the expression of three bovine oocyte expressed long non-coding RNAs (lncRNAs), OOSNCR1, OOSNCR2, and OOSNCR3, was characterized in somatic tissues, the ovarian follicle, and throughout early embryonic development. Moreover, the functional requirement of each transcript during oocyte maturation and early embryonic development was investigated using a siRNA-mediated knockdown approach. Tissue distribution analysis revealed that OOSNCR1, OOSNCR2 and OOSNCR3 are predominantly expressed in fetal ovaries. Follicular cell expression analysis revealed that these lncRNAs are highly expressed in the oocytes, with minor expression detected in the cumulus cells (CCs) and mural granulosa cells (mGCs). The expression for all three genes was highest during oocyte maturation, decreased at fertilization, and ceased altogether by the 16-cell stage. Knockdown of OOSNCR1, OOSNCR2 and OOSNCR3 in immature oocytes was achieved by microinjection of the cumulus-enclosed germinal vesicle (GV) oocytes with siRNAs targeting these lncRNAs. Knockdown of OOSNCR1, OOSNCR2 and OOSNCR3 did not affect cumulus expansion, but oocyte survival at 12 h post-insemination was significantly reduced. In addition, knockdown of OOSNCR1, OOSNCR2 and OOSNCR3 in immature oocytes resulted in a decreased rate of blastocyst development, and reduced expression of genes associated with oocyte competency such as nucleoplasmin 2 (NPM2), growth differentiation factor 9 (GDF9), bone morphogenetic protein 15 (BMP15), and JY-1 in MII oocytes. The data herein suggest a functional requirement of OOSNCR1, OOSNCR2, and OOSNCR3 during bovine oocyte maturation and early embryogenesis., Competing Interests: Declaration of Competing Interest The authors declare no conflicts of interest., (Copyright © 2024 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier B.V. All rights reserved.)

Research Question: Is there a relationship between the pronuclear axis and the first cleavage plane formation in human pronuclear-stage embryos, and what are the effects on ploidy and clinical pregnancy rates?, Design: Transferred embryos were followed up until their prognoses. A total of 762 embryos formed two cells and reached the blastocyst stage after normal fertilization in a time-lapse incubator. Embryos were classified into three groups: group A: embryos in which the first plane of division was formed parallel to the axis of the pronucleus; group B: embryos in which cases of oblique formation were observed; and group C: embryos in which cases of perpendicular formation were observed., Results: The euploidy rate was significantly higher in groups A and B than those in group C (P < 0.01), whereas the aneuploidy rate was significantly higher in group C (P < 0.01) than in groups A and B. No differences were found between the three groups in frequency of positive HCG-based pregnancy tests, frequency of clinical pregnancies, miscarriage rates or delivery rates., Conclusions: The formation pattern of the first plane of division relative to the pronuclear axis was a predictor of embryonic ploidy, with a reduced rate of euploidy and a high probability of aneuploidy observed when the first plane of division was perpendicular to the pronuclear axis., (Copyright © 2024 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)

Ants are one of the most ecologically and evolutionarily successful groups of animals and exhibit a remarkable degree of phenotypic diversity. This success is largely attributed to the fact that all ants are eusocial and live in colonies with a reproductive division of labor between morphologically distinct queen and worker castes. Yet, despite over a century of studies on caste determination and evolution in ants, we lack a complete ontogenetic series from egg to adult for any ant species. We, therefore, present a developmental table for the Pharaoh ant Monomorium pharaonis, a species whose colonies simultaneously produce reproductive queens and completely sterile workers. In total, M. pharaonis embryonic, larval, and pupal development lasts 45 days. During embryogenesis, the majority of developmental events are conserved between M. pharaonis and the fruit fly Drosophila melanogaster. We discovered, however, two types of same-stage embryos before gastrulation: (1) embryos with internalized germ cells; and (2) embryos with germ cells outside of the blastoderm at the posterior pole. Moreover, we also found two-types of embryos following germ band extension: (1) embryos with primordial germ cells that will develop into reproductive queens; and (2) embryos with no germ cells that will develop into completely sterile workers. Together, these data show that queen and worker castes are already determined and differentiated by early embryogenesis. During larval development, we confirmed that reproductive and worker larvae proceed through three larval instars. Using anatomical and developmental markers, we can further discern the development of gyne (unmated queen) larvae, male larvae, and worker larvae as early as the 1st instar. Overall, we hope that the ontogenetic series we present here will serve as a blueprint for the generation of future ant developmental tables., (© 2024 The Author(s). Journal of Experimental Zoology Part B: Molecular and Developmental Evolution published by Wiley Periodicals LLC.)

Background: Our study aimed to investigate the embryological characteristics and clinical outcomes of oocytes with indented zona pellucida (iZP)., Methods: Cases with all oocytes containing iZP were compared with subjects whose oocytes presented morphologically normal ZP (nZP, controls), regarding embryological and clinical outcomes. Cases consisted of 56 subjects that underwent 109 assisted reproductive treatment (ART) cycles, and controls were made of 574 patients that were enrolled in 1095 ART cycles., Results: Patients with iZP presented a significantly prolonged duration of infertility and were more likely to have primary infertility. Regarding embryonic development, cases with iZP evidenced significantly lower rates of retrieved oocytes, oocyte maturity, fertilisation, embryo cleavage, high-quality embryo and blastocyst ( p  < .05). Relatively to clinical outcomes, 32 patients with iZP underwent fresh embryo transfer. Compared to controls, the rates of clinical pregnancy (CP) (43.8% vs. 65.9%, p  = .014) and live-birth delivery (LBD) (34.4% vs. 58.5%, p  = .009) were significantly lower. No significant differences were observed between groups regarding clinical outcomes after frozen-thawed embryo transfer (FET). However, cumulative rates elicited excellent CP (49.3%) and LBD (42.0%) rates in cases with iZP. In cases where rescue-intracytoplasmic sperm injection (ICSI) was needed, cases with iZP could achieve lower but nevertheless high cumulative CP (52.6%) and LBD (36.8%) rates., Conclusions: Patients with iZP present satisfactory embryological and clinical outcomes, with ICSI and FET cycles improving the global outcomes.

Knowledge of embryonic body posture as a reflection of neurodevelopment is limited. Here, we investigate embryonic body posture, preference, symmetry, and lateralization during periods of rest using virtual reality (VR) and four-dimensional (4D) ultrasound techniques to enhance our knowledge of neurodevelopment. It is hypothesized that lateralization and asymmetry in embryonic posture are absent. Embryos from low-risk pregnancies at 9 weeks gestational age (GA) were analyzed using a 30-min 4D ultrasound and VR techniques. In the middle of an embryonic resting period, the total body posture was determined by combining joint positions. Interindividual and intraindividual postures were analyzed over time to identify posture preferences, asymmetry, and lateralization. Sixteen embryos (N = 210 middle frames out of 210 resting periods) displayed 27 different embryonic body postures. Interindividual and intraindividual variations in postures were seen. Asymmetry was noted in upper extremities, whereas lateralization of the head and spine was observed in nine embryos. Intraindividual and interindividual variation in embryonic posture at 9 weeks' GA is seen, suggesting the presence of supraspinal influences on body posture this early. Future interdisciplinary longitudinal studies on embryonic posture may provide further insights into embryonic central nervous system development, contributing to our understanding of embryonic health., (© 2024 The Author(s). Developmental Psychobiology published by Wiley Periodicals LLC.)

Extracellular vesicles, also known as exosomes, influence numerous cellular functions by regulating different signaling pathways. However, their role in animal reproduction remains understudied. This study aimed to evaluate the effects of porcine follicular fluid-derived exosomes (pff-Exos) on porcine oocyte in vitro maturation and parthenogenetic embryo development. We obtained pff-Exos through mixed-method ultracentrifugation and size-exclusion chromatography. Transmission electron microscopy revealed an increase in the expression of exosome markers in the first four of thirteen fractions. The number of pff-Exo was 2.2 × 10 6 particles per microliter. The highest maturation rate of porcine oocytes treated with pff-Exo was observed with 1.1 × 10 7 particles of pff-Exo in the absence of porcine follicular fluid (pFF) culture conditions. Moreover, increased expression of Gdf9 and Bmp15 was observed. The developmental rate was the highest upon treatment with 1.1 × 10 7 particles of pff-Exo, which increased the total cell number in blastocysts. Embryonic development to the 2-cell stage was similar between the control and pff-Exo groups; however, development to the 4-cell stage and blastocyst was significantly increased in the pff-Exo group (61.6 ± 6.08 % and 29.72 ± 1.41 %, respectively; P < 0.05) compared with that in the control group (42.0 ± 5.19 % and 18.14 ± 1.78 %, respectively). The expression levels of Oct4, Sox2, Bcl2, Elf4, and Gcn5 significantly increased at the pff-Exo 2-cell stage, whereas those of Bax, Hdac1, Hdac6, and Sirt6 decreased. Specifically, the Oct4, Sox2, Elf4, Gcn5, and Hdac6 levels remained stable in pff-Exo 4-cell embryos, whereas those of p53 and Hat1 were reduced and increased, respectively. Treatment with pffExos significantly increased H3K9 and H3K14 acetylation levels. These results demonstrate that pff-Exo affects the in vitro maturation of porcine oocytes and early embryonic development by regulating gene expression., Competing Interests: Declaration of competing interest The authors have no conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Monozygotic (MZ) twins are believed to arise from the fission of a single fertilized embryo at different stages. Monochorionic MZ twins, who share one chorion, originate from the splitting of the inner cell mass (ICM) within a single blastocyst. In the classic model for dichorionic MZ twins, the embryo splits before compaction, developing into two blastocysts. However, there are a growing number of ART cases where a single blastocyst transfer results in dichorionic MZ twins, indicating that embryo splitting may occur even after blastocyst formation., Objective and Rationale: For monochorionic MZ twins, we conducted a comprehensive analysis of the cellular mechanisms involved in ICM splitting, drawing from both ART cases and animal experiments. In addition, we critically re-examine the classic early splitting model for dichorionic MZ twins. We explore cellular mechanisms leading to two separated blastocysts in ART, potentially causing dichorionic MZ twins., Search Methods: Relevant studies including research articles, reviews, and conference papers were searched in the PubMed database. Cases of MZ twins from IVF clinics were found by using combinations of terms including 'monozygotic twins' with 'IVF case report', 'ART', 'single embryo transfer', or 'dichorionic'. The papers retrieved were categorized based on the implicated mechanisms or as those with unexplained mechanisms. Animal experiments relating to MZ twins were found using 'mouse embryo monozygotic twins', 'mouse 8-shaped hatching', 'zebrafish janus mutant', and 'nine-banded armadillo embryo', along with literature collected through day-to-day reading. The search was limited to articles in English, with no restrictions on publication date or species., Outcomes: For monochorionic MZ twins, ART cases and mouse experiments demonstrate evidence that a looser ICM in blastocysts has an increased chance of ICM separation. Physical forces facilitated by blastocoel formation or 8-shaped hatching are exerted on the ICM, resulting in monochorionic MZ twins. For dichorionic MZ twins, the classic model resembles artificial cloning of mouse embryos in vitro, requiring strictly controlled splitting forces, re-joining prevention, and proper aggregation, which allows the formation of two separate human blastocysts under physiological circumstances. In contrast, ART procedures involving the transfer of a single blastocysts after atypical hatching or vitrified-warmed cycles might lead to blastocyst separation. Differences in morphology, molecular mechanisms, and timing across various animal model systems for MZ twinning can impede this research field. As discussed in future directions, recent developments of innovative in vitro models of human embryos may offer promising avenues for providing fundamental novel insights into the cellular mechanisms of MZ twinning during human embryogenesis., Wider Implications: Twin pregnancies pose high risks to both the fetuses and the mother. While single embryo transfer is commonly employed to prevent dizygotic twin pregnancies in ART, it cannot prevent the occurrence of MZ twins. Drawing from our understanding of the cellular mechanisms underlying monochorionic and dichorionic MZ twinning, along with insights into the genetic mechanisms, could enable improved prediction, prevention, and even intervention strategies during ART procedures., Registraiton Number: N/A., (© The Author(s) 2024. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology.)