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The transition from a highly subjective morphological assessment to time-lapse imaging improves the accuracy of predicting embryonic developmental potential. In actual operations, embryos are cultured for 2–3 days in a time-lapse monitoring system before being transferred to recipients. However, most existing prediction models require videos or images spanning a five-day period. Therefore, it is necessary to develop a method that accurately predicts blastocyst formation given input data spanning only 2–3 days. In this study, we propose a method for predicting blastocyst formation using early morphokinetic and morphological parameters prior to the five-cell stage. We employed a YOLOv5 pretrained deep-learning network to recognize the first four-cell stages for the accurate extraction of morphokinetic parameters and used these parameters as inputs to construct four long short-term memory-based morphokinetic models for blastocyst formation prediction, obtaining the best area-under-the-curve (AUC) value of 0.7297 [0.669–0.884]. We then extracted the three frames before and after the t1–t4 time points and calculated the image entropy and gray-level co-occurrence matrix entropy as morphological features to build a prediction model. This model was subsequently fused with the morphokinetic model, and an AUC of 0.8325 [0.7601–0.9067] was achieved. Our results have implications for automatic embryo screening given information on early embryonic development. [ABSTRACT FROM AUTHOR]

We use molecular dynamics simulations to examine the homogeneous nucleation of ice VII from metastable liquid water. An unsupervised machine learning classification identifies two distinct local structures composing Ice VII nuclei. The seeding method, combined with the classical nucleation theory (CNT), predicts the solid–liquid interfacial free energy, consistent with the value from the mold integration method. Meanwhile, the nucleation rates estimated from the CNT framework and brute force spontaneous nucleations are inconsistent, and we discuss the reasons for this discrepancy. Structural and dynamical heterogeneities suggest that the potential birthplace for an ice VII embryo is relatively ordered, although not necessarily relatively immobile. Moreover, we demonstrate that without the formation of hydrogen-bond links, ice VII embryos do not grow. [ABSTRACT FROM AUTHOR]

Background: Early seed germination in crops can confer a competitive advantage against weeds and reduce the time to maturation and harvest. WRKY transcription factors regulate many aspects of plant development including seed dormancy and germination. Both positive and negative regulators of seed germination have been reported in many plants such as rice and Arabidopsis. Using a transient expression system, we previously demonstrated that OsWRKY71 is a negative regulator of gibberellin (GA) signaling in aleurone cells and likely forms a "repressosome" complex with other transcriptional repressors. Hence, it has the potential to impact seed germination properties. Results: In this study, we demonstrate that OsWRKY71, a Group IIa WRKY gene, appeared at the same time as seed-bearing plants. Rice mutants lacking OsWRKY71 have seeds and embryos that germinate earlier than wildtype controls. In oswrky71 aleurone layers, α-amylase activity was hypersensitive to stimulation by GA3 and hyposensitive to inhibition by abscisic acid (ABA). Early germination in oswrky71 intact seeds was also hyposensitive to ABA. Transcriptomic profiling during embryo germination and early post-germination growth demonstrates that OsWRKY71 influences the expression of 9–17% of genes in dry and imbibing embryos. Compared to wildtype embryos, the mutant transcriptomes have large temporal shifts at 4, 8 and 12 h after imbibition (HAI). Importantly, many genes involved in the ABA-dependent inhibition of seed germination were downregulated in oswrky71-1. This mutant also displayed altered expression of multiple ABA receptors (OsPYLs/RCARs) that control ABA signaling and the VP1-SDR4-DOG1L branch of ABA signaling that promotes seed dormancy. Association studies reveal an OsWRKY71-containing quantitative trait locus involved in low-temperature seed germinability, qLTG-2. Indeed, oswrky71 seeds germinated early at 15 °C. Conclusions: Rice Group-IIa WRKY transcription factor OsWRKY71 is a master regulator of germination that influences the expression of 9–17% of genes in dry and imbibing embryos. It is also most likely the primary candidate of low-temperature seed germinability QTL, qLTG-2. We propose that knockouts of OsWRKY71 can generate rice varieties with improved germination properties under normal or low-temperature conditions. [ABSTRACT FROM AUTHOR]

The invasion of terrestrial ecosystems by tetrapods (c. 375 million years [Ma]) represents one of the major evolutionary transitions in the history of life on Earth. The success of tetrapods on land is linked to evolutionary novelties. Among these, the evolution of a tympanic ear contributed to mitigating the problem of an impedance mismatch between the air and the fluid embedding sound-detecting hair cells in the inner ear. 1,2,3 Pioneering studies advocated that similarities in the tympanic ear of tetrapods could only result from a single origin of this structure in the group, 4,5 an idea later challenged by paleontological and developmental data. 4,6,7,8 Current evidence suggests that this sensory structure evolved independently in amphibians, mammals, and reptiles, 1,6 but it remains uncertain how many times tympanic hearing originated in crown reptiles. 9,10 We combine developmental information with paleontological data to evaluate the evolution of the tympanic ear in reptiles from two complementary perspectives. Phylogenetically informed ancestral reconstruction analyses of a taxonomically broad sample of early reptiles point to the presence of a tympanic membrane as the ancestral condition of the crown group. Consistently, comparative analyses using embryos of lizards and crocodylians reveal similarities, including the formation of the tympanic membrane within the second pharyngeal arch, which has been previously reported for birds. Therefore, both our developmental and paleontological data suggest a single origin for the tympanic middle ear in the group, challenging the current paradigm of multiple acquisitions of tympanic hearing in living reptiles. [Display omitted] • Fossils reveal that the presence of tympanic membrane is ancestral to crown reptiles • The eardrum of lizards and crocodylians develops within the second pharyngeal arch • Fossils and embryos suggest a single origin of the tympanic ear in crown reptiles The tympanic ear is a key trait related to the evolutionary success of tetrapods on land, as it enhances the hearing capabilities of animals in this environment. Bronzati et al. use data from embryonic development and from the fossil record and provide evidence for a single origin of tympanic hearing in the group of living reptiles. [ABSTRACT FROM AUTHOR]

Skeletal muscle formation involves tight interactions between muscle cells and associated connective tissue fibroblasts. Every muscle displays the same type of organisation, they are innervated in the middle and attached at both extremities to tendons. Myonuclei are heterogeneous along myotubes and regionalised according to these middle and tip domains. During development, as soon as myotubes are formed, myonuclei at muscle tips facing developing tendons display their own molecular program. In addition to molecular heterogeneity, a subset of tip myonuclei has a fibroblastic origin different to the classical somitic origin, highlighting a cellular heterogeneity of myonuclei in foetal myotubes. To gain insights on the functional relevance of myonucleus heterogeneity during limb development, we used 2D culture and co-culture systems to dissociate autonomous processes (occurring in 2D-cultures) from 3D-environment of tissue development. We also assessed the role of muscle contraction in myonucleus heterogeneity in paralysed limb muscles. The regionalisation of cellular heterogeneity was not observed in 2D cell culture systems and paralyzed muscles. The molecular signature of MTJ myonuclei was lost in a dish and paralysed muscles indicating a requirement of 3D-enviroment and muscle contraction for MTJ formation. Tip genes that maintain a regionalized expression at myotube tips in cultures are linked to sarcomeres. The behaviour of regionalized markers in cultured myotubes and paralyzed muscles allows us to speculate whether the genes intervene in myogenesis, myotube attachment or MTJ formation. Highlights: • The molecular signature of MTJ myonuclei is lost in cultured myotubes and paralysed muscles • Genes expressed in muscle tips that maintain their regionalised expression in cultured myotubes are linked to sarcomeric proteins • Cellular heterogeneity of myonuclei is observed in cultured myotubes but with no regionalisation • BMP signalling regulates fibroblast nucleus incorporation into cultured myotubes [ABSTRACT FROM AUTHOR]

Avian species are essential resources for human society, with their preservation and utilization heavily dependent on primordial germ cells (PGCs). However, efficient methods for isolating live PGCs from embryos remain elusive in avian species beyond chickens, and even in chickens, existing techniques have shown limited efficiency. In this study, we present a rapid, simple, and cost‐effective method for labeling and sorting circulating‐stage PGCs across various avian species, including Carinatae and Ratitae, using Lycopersicon Esculentum (Tomato) lectin (LEL). Notably, this method demonstrates high sorting efficiency by identifying a wide range of PGC subtypes while preserving the proliferative and migratory potential of chicken PGCs. This approach is anticipated to significantly contribute to the conservation, research, and agricultural industries related to avian species globally. [ABSTRACT FROM AUTHOR]

The droplet microarray (DMA) platform is a powerful tool for high‐throughput biological and chemical applications, enabling miniaturization and parallelization of experimental processes. Capable of holding hundreds of nanoliter droplets, it facilitates the screening and analysis of samples, such as cells, bacteria, embryos, and spheroids. Handling thousands of small volumes in parallel presents significant challenges. In this study, we utilize the open format of the DMA for controlled, parallel high‐throughput liquid manipulations using the sandwich technique. We demonstrate high‐throughput medium replacement at nanoliter‐scale, maintaining high cell viability on DMA for up to 7 days; for HeLa‐CLL2 cells (adherent) and SU‐DHL4 cells (suspension), and up to 14 days for HEK293 spheroids. Additionally, we achieve highly parallel aliquot uptake from nanoliter droplets, enabling non‐destructive cell viability assessments. Furthermore, the presented method enables the parallel transfer of cell spheroids between different DMAs, allowing transfer and pooling of spheroids in seconds without damage. These advances significantly enhance the capabilities of the DMA platform, enabling long‐term cell culture in nanoliter droplets and parallel sampling for high‐throughput cell or spheroid manipulation. This broadens the scope of DMA's potential applications in fields such as cell‐based high‐throughput screening, formation of complex 3D cell models for drug screening, and microtissue engineering. [ABSTRACT FROM AUTHOR]

Medical providers have an ethical duty to disclose clinically significant errors involving gametes and embryos. Although not mandatory, disclosure of errors causing no harm or near misses is recommended. In addition, clinics should have written policies in place for reducing and disclosing errors. [ABSTRACT FROM AUTHOR]

The current article provides an ethical reflection on the moral status of the human embryo, which is a crucial factor in determining permissible actions involving embryos and the extent of their protection. It advocates for the extension of the research period for embryos to 28-days post fertilization. It also states that integrated embryo-like structures (ELSs) should not currently be given the same moral status as natural embryos. However, if they pass the relevant tests, they should be subject to the same rules as natural embryos. [ABSTRACT FROM AUTHOR]

Objectives: Photoangiolytic lasers have yielded significant innovation in laryngeal surgery in the last 25 years. After the discontinuation of the potassium titanyl phosphate (KTP) laser, a novel 445-nm blue laser was developed. The optimal balance between a laser's desired tissue effects and collateral tissue damage is a major determinant of laser selection in microlaryngeal surgery. The shell-less incubation system for the chick chorioallantoic membrane (CAM) simulates the microvasculature of the human vocal fold and is useful for testing effects of laser settings and in simulated surgery. The aim of this study is to compare the tissue effects of the KTP and blue lasers using the shell-less CAM model. Methods: The shell-less incubation system contains: polymethylpentene film (used as a culture vessel), calcium lactate and distilled water supplementations. By using this system, the chick chorioallantoic membrane (CAM) can be fully exposed with a good field for surgery simulation. The effects of the 2 lasers (532 nm KTP and 445 nm blue) were quantified at clinically relevant energy settings and laser distances from target. Measures included imaging real-time vascular reactions in the CAM model, post-procedure histologic analysis of CAM tissue and temperature changes. Results: Vessel coagulation and rupture rates were less common with the blue laser compared with the KTP laser. Histologic analysis demonstrated less tissue disruption with the blue laser. Temperature changes were less with the blue laser. Conclusion: In this CAM model with specific conditions, the blue laser reveals less tissue damage than the KTP laser. Suitable working distance and power setting of the laser are necessary for desired tissue effects. Level of Evidence: Level 3. [ABSTRACT FROM AUTHOR]

Diapause is a survival strategy for freshwater crustaceans in the genus Daphnia that involves a genetically encoded inhibition of organism growth, development and reproduction. While the environmental triggers for Daphnia to produce diapause‐destined embryos and those that induce hatching are well documented, the influence of environmental challenges during different diapause sub‐phases remains unexplored.In this study, we exposed diapause‐destined embryos of Daphnia magna to different temperatures and light conditions during designated diapause sub‐phases.Our results underscore the importance of the maternal environment for the embryos that are in preparation for diapause. Moreover, we demonstrate a period of obligate developmental arrest during which reinitiation of development cannot be triggered. Additionally, we illustrate the embryos' ability to adjust hatching in response to environmental changes during the hatching process.This study reveals the inherent developmental pattern in Daphnia embryos as they progress through diapause and their adaptability to environmental changes. [ABSTRACT FROM AUTHOR]

Background & Objective: Polycystic ovary syndrome (PCOS) is one of the most common causes of infertility in women worldwide. In vitro fertilization (IVF) is a method used to increase the chance of fertilization. This study was conducted with aim to assess the factors associated with IVF success in women with PCOS. Materials & Methods: This retrospective study was performed on women aged 18 to 46 years with PCOS undergoing IVF in the Infertility Research and Clinical Center, Yazd, Iran, from April 2016 to February 2017. The success of IVF was evaluated in two stages with a β-hCG blood test and ultrasound imaging. The variables of age, number of total embryos, quality of embryos, fertilization rate, number of transferred embryos, and endometrial thickness were measured. Results: In this study, 600 women were included. Of these, 213 and 144 patients had positive pregnancy by β-hCG blood test and ultrasound imaging, respectively. The number of total embryos (OR=1.88) and A-quality embryos (OR=1.81) were significantly associated with positive β-hCG test. Regarding pregnancy confirmation by ultrasound imaging, A-quality embryo (OR=3.75), and B-quality embryo (OR=2.68) had a significant relationship with fertilization (OR=8.63). Conclusion: Age, the quality of embryos and fertilization rate can predict IVF success in PCOS patients. However, no significant relationship was found between endometrial thickness and the number of transferred embryos and pregnancy response. Further studies are needed to obtain more understanding in this regard. [ABSTRACT FROM AUTHOR]

[Display omitted] [ABSTRACT FROM AUTHOR]

Brown algae are multicellular photosynthetic organisms that have evolved independently of plants and other algae. Here, we have studied the determinism of body axis formation in the kelp Saccharina latissima. After microdissection of the embryo, we show that the stalk, an empty cell that retains the embryo on the maternal tissue, represses longitudinal cell divisions in the early embryo, thereby reinforcing the establishment of the initial apico-basal axis. In addition, it promotes cell growth and controls cell shape and arrangement in the flat oblong embryo composed of cells aligned in rows and columns. Although the stalk persists for several weeks until the embryo reaches at least 500 cells, proper embryogenesis requires connection to maternal tissue only during the first 4 days after fertilisation, i.e. before the embryo reaches the 8-cell stage. Transplantation experiments indicate that the maternal signal is not diffused in seawater, but requires contact between the embryo and the maternal tissue. This first global quantitative study of brown algal embryogenesis highlights the role of MUM, an unknown maternal message, in the control of growth axes and tissue patterning in kelp embryos. [ABSTRACT FROM AUTHOR]

Summary: During Arabidopsis embryogenesis, the transition of the embryo's symmetry from radial to bilateral between the globular and heart stage is a crucial event, involving the formation of cotyledon primordia and concurrently the establishment of a shoot apical meristem (SAM). However, a coherent framework of how this transition is achieved remains to be elucidated.In this study, we investigated the function of DELAYED GREENING 1 (DG1) in Arabidopsis embryogenesis using a newly identified dg1‐3 mutant. The absence of chloroplast‐localized DG1 in the mutants led to embryos being arrested at the globular or heart stage, accompanied by an expansion of WUSCHEL (WUS) and SHOOT MERISTEMLESS (STM) expression. This finding pinpoints the essential role of DG1 in regulating the transition to bilateral symmetry. Furthermore, we showed that this regulation of DG1 may not depend on its role in plastid RNA editing.Nevertheless, we demonstrated that the DG1 function in establishing bilateral symmetry is genetically mediated by GENOMES UNCOUPLED 1 (GUN1), which represses the transition process in dg1‐3 embryos.Collectively, our results reveal that DG1 functionally antagonizes GUN1 to promote the transition of the Arabidopsis embryo's symmetry from radial to bilateral and highlight the role of plastid signals in regulating pattern formation during plant embryogenesis. [ABSTRACT FROM AUTHOR]

Background: Spermidine (SPD) is an intermediate compound in the polyamine metabolism which takes critical part in a variety of cellular processes. In particular, it has been reported to exert anti-aging effects, suppress the age-related diseases, and extend lifespan across species. However, whether it has the favorable influence on the quality of postovulatory aged oocytes remains elusive. Methods: Immunostaining and fluorescence intensity measurement were used to evaluate the effects of postovulatory aging and SPD supplementation on the oocyte fragmentation, spindle/chromosome structure, actin polymerization, dynamics of cortical granules (CGs) and ovastacin, mitochondrial distribution and function, as well as autophagy levels. In addition, in vitro sperm binding assay and in vitro fertilization (IVF) experiment were applied to assess the impacts of postovulatory aging and SPD supplementation on the sperm binding ability and fertilization capacity of oocytes. Results: Here, we showed that supplementation of SPD during postovulatory aging could relieve the deterioration of porcine oocytes. Specifically, we found that postovulatory aging impaired the oocyte quality by damaging the morphological integrity of oocytes, maintenance of spindle/chromosome structure, and dynamics of actin cytoskeleton. Postovulatory aging also weakened the sperm binding ability and fertilization capacity of oocytes by compromising the distribution pattern of CGs and their content ovastacin. Notably, supplementation of SPD attenuated these defects in postovulatory aged porcine oocytes via strengthening mitochondrial function, eliminating excessive reactive oxygen species (ROS), inhibiting apoptosis, and enhancing autophagy levels. Conclusion: Altogether, our findings demonstrate that SPD supplementation is a feasible approach to ameliorate the quality of postovulatory aged oocytes, which can be potentially applied to the human assisted reproductive technology (ART) and in vitro production of animal embryos. [ABSTRACT FROM AUTHOR]

The translational repressor Nanos (Nos) regulates a single target, maternal hunchback (hb) mRNA, to govern abdominal segmentation in the early Drosophila embryo. Nos is recruited to sites in the 3′ UTR of hb mRNA in collaboration with the sequence-specific RNA-binding protein Pumilio (Pum); on its own, Nos has no binding specificity. Nos is expressed at other stages of development, but very few mRNA targets that might mediate its action at these stages have been described. Nor has it been clear whether Nos is targeted to other mRNAs in concert with Pum or via other mechanisms. In this report, we identify mRNAs targeted by Nos via 2 approaches. First, we identify mRNAs depleted upon expression of a chimera bearing Nos fused to the nonsense mediated decay (NMD) factor Upf1. We find that, in addition to hb, Upf1-Nos depletes approximately 2,600 mRNAs from the maternal transcriptome in early embryos. Virtually all of these appear to be targeted in a canonical, hb-like manner in concert with Pum. In a second, more conventional approach, we identify mRNAs that are stabilized during the maternal zygotic transition (MZT) in embryos from nos- females. Most (86%) of the 1,185 mRNAs regulated by Nos are also targeted by Upf1-Nos, validating use of the chimera. Previous work has shown that 60% of the maternal transcriptome is degraded in early embryos. We find that maternal mRNAs targeted by Upf1-Nos are hypoadenylated and inefficiently translated at the ovary–embryo transition; they are subsequently degraded in the early embryo, accounting for 59% of all destabilized maternal mRNAs. We suggest that the late ovarian burst of Nos represses a large fraction of the maternal transcriptome, priming it for later degradation by other factors in the embryo. The translational repressor Nanos regulates abdominal segmentation in the early Drosophila embryo, but it remains unclear what are the mRNAs targets that regulate its function. This study identifies extensive regulation of maternal mRNAs by Nanos during the transition from ovarian to embryonic development in Drosophila. [ABSTRACT FROM AUTHOR]

Anther and microspore cultures are efficient methods for inducing haploids in plants. The microspore culture by chromosome-doubling method can produce double haploid lines, developing pure lines within the first or second generations. This study aimed to induce haploid plants in Platycodon grandiflorum using the shed-microspore culture method. P. grandiflorum floral buds (n = 1503) were cultured in six types of medium to induce haploids. Anthers were placed on a solid–liquid double-layer medium and cold pre-treated at 9 °C for one week, followed by incubation in the dark at 25 °C. Embryogenesis was observed after approximately 70 days of culture, producing haploid plants through regeneration. Of the 1503 floral buds, embryos developed in 120 buds, resulting in the induction of 402 individuals. Among the media used, Schenk and Hildebrandt (SH) and 1/2SH exhibited high efficiency, with embryogenesis ratios of 12% and 13.4%, respectively. Additionally, the highest embryogenesis ratio (15.3%) was observed in flower buds sized 10 mm or less. Therefore, we established shed-microspore culture conditions to induce haploids in P. grandiflorum. Using this method, haploids can be efficiently induced in P. grandiflorum, shortening the breeding period by enabling the rapid development of inbred lines. [ABSTRACT FROM AUTHOR]

Limited evidence suggests that variation in phenotypic plasticity within populations may arise largely from environmental sources, thereby constraining its evolvability. This is of concern for temperature-sensitive metabolism in the face of climate change. We quantified the relative influence of the developmental environment versus genes on the metabolic plasticity of avian embryos to temperature. We partially cross-fostered 602 house sparrow eggs (Passer domesticus), measured the heart rate plasticity of these embryos to egg temperature and partitioned variance in plasticity. We found that the foster (incubation) environment was the sole meaningful source of variance in embryonic plasticity (not genes, pre-laying effects or ambient conditions). In contrast to heart rate plasticity, offspring growth was influenced by the foster environment, genes/pre-laying parental effects and ambient conditions. Although embryonic plasticity to temperature varied in this population, these results suggest that it is unlikely to evolve quickly. Nevertheless, the expression of this plasticity may be able to shift between generations in response to changes in the developmental environment. Whether the multidimensional plasticity of heart rate to both current temperature and the developmental environment is itself an adaptive, evolved trait allowing avian embryos to optimize their metabolic plasticity to their current environment remains to be tested. [ABSTRACT FROM AUTHOR]

The birth of a baby or the hatching of an egg are moments when the risk of predation is particularly high. In oviparous species where clutches contain many eggs, one strategy for limiting predation is based on mechanisms that synchronise hatching, which reduces the individual risk of being targeted and may allow hatchlings to emerge under parental protection. These mechanisms involve communication signals between eggs and between eggs and parents. While previous studies in various species have shown the importance of sound and vibration signals in these exchanges, how embryos integrate this information remains poorly understood. Here we show that crocodile embryos respond differently to calls emitted by siblings (hatching calls) and to vibrations mimicking parental intervention (rubbing and scratching the shell) or the presence of a predator, suggesting that these two stimuli carry different information. Through playback experiments, we confirm that hatching calls elicit vocal responses and synchronous hatching in mature embryos, while embryos hatch earlier when they perceive vibrations. Our study underlines that the control of hatching by external factors can rely on differential apprehension of multiple sensory inputs by embryos. [ABSTRACT FROM AUTHOR]

(1) Background: Does the variation in sequential development times of embryos, observed through time-lapse monitoring, between the two study groups play a role in predicting pregnancy success? (2) Methods: The prospective double-arm study was to identify the morphokinetic parameters specific to embryos that were capable of implanting and were conducted on 89 embryos cultured in the Esco Miri time-lapse incubator, divided into two groups: Lot A, consisting of 57 embryos that successfully implanted and resulted in life birth rate (LBR), and Lot B (NLB), comprising 32 embryos that did not implant, leading to a negative beta-hCG outcome. (3) Results: Baseline characteristics, including female age, were not found to be statistically significant (p > 0.01). In contrast, there is a highly statistically significant difference concerning oocytes (p = 0.0029). Morphokinetic variables represented by sequential culture times were not statistically significant (p > 0.01) when comparing the two groups. However, the negative mean differences between these parameters suggest that the times for Lot A are better (shorter) than those for Lot B. While not statistically significant, these differences may still have practical significance. In the case of grading, the difference is considered to be extremely statistically significant (p < 0.01). (4) Conclusions: Although there are no statistically significant differences in sequential timings (p > 0.01) between the two groups, there are parameters indicating predictive potential for exploring pregnancy in embryo morphokinetics. [ABSTRACT FROM AUTHOR]

Vertebrate embryos are protected from bacterial infection by various maternally derived factors, yet little is known about the defence mechanisms in elasmobranchs. This study aimed to characterize the intracapsular environment of freshly laid eggs of the oviparous catshark (Scyliorhinus torazame) by investigating the microbial abundance and microbiota to understand its potential contribution to embryonic defence. The egg capsule of oviparous elasmobranchs is tightly sealed until pre‐hatching, after which seawater flows into the capsule, exposing the embryos to the surrounding seawater. We found that early embryos were highly vulnerable to environmental pathogens, suggesting that the embryos are somehow protected from infection before pre‐hatching. Indeed, the intracapsular environment of freshly laid eggs exhibited significantly low bacterial density, maintained until pre‐hatching. Furthermore, the microbiome inside eggs just after oviposition differed markedly from those of rearing seawater and adult oviducal gland epithelia; these eggs were predominantly populated by an unidentified genus of Sphingomonadaceae. Overall, this study provides compelling evidence that early embryos of oviparous cloudy catshark are incubated in a clean intracapsular environment that potentially plays a significant role in embryonic development in oviparous elasmobranchs. [ABSTRACT FROM AUTHOR]

Pharyngeal endoderm cells undergo convergence and extension (C&E), which is essential for endoderm pouch formation and craniofacial development. Our previous work implicates Gα13/RhoAmediated signaling in regulating this process, but the underlying mechanisms remain unclear. Here, we have used endoderm-specific transgenic and Gα13mutant zebrafish to demonstrate that Gα13 plays a crucial role in pharyngeal endodermC&E by regulating RhoA activation and E-cadherin expression. We showed that during C&E, endodermal cells gradually establish stable cell-cell contacts, acquire apical-basal polarity and undergo actomyosin-driven apical constriction, which are processes that require Gα13. Additionally, we found that Gα13-deficient embryos exhibit reduced E-cadherin expression, partially contributing to endoderm C&E defects. Notably, interfering with RhoA function disrupts spatial actomyosin activation without affecting E-cadherin expression. Collectively, our findings identify crucial cellular processes for pharyngeal endoderm C&E and reveal that Gα13 controls this through two independent pathways - modulating RhoA activation and regulating E-cadherin expression - thus unveiling intricate mechanisms governing pharyngeal endoderm morphogenesis. [ABSTRACT FROM AUTHOR]

Context • The early symptoms of embryo development arrest are not typical. There is currently no model tool available to predict embryo development arrest. Objective • To explore the influencing factors of embryo development arrest in early pregnancy and build a risk prediction model. Methods • From May 2019 to March 2023, 277 patients suspected of embryonic development arrest during the first ultrasound examination in the Department of Obstetrics and Gynecology of the Ninth Affiliated Hospital of Soochow University were retrospectively selected as the study subjects. They were divided into diapause group and non-diapause group according to the second ultrasound (review after 1-2 weeks) to diagnose whether embryo development arrest. Collect two sets of data for analysis, Screen out the influencing factors of early pregnancy embryo development arrest. The logistic regression model and random forest model were constructed respectively. Evaluate the predictive performance of two statistical models. Results • Out of 277 suspected cases of embryonic developmental arrest, 88 were ultimately confirmed. Older age (OR: 2.259, P = .017), higher ultrasonic blood flow resistance index (RI) (OR: 1.728, P = .038), higher ultrasonic gestational sac diameter/embryo head hip length ratio (MSD/CRL) (OR:1.919, P = .007), lower progesterone (OR: 0.562, P = .011), and lower pregnancy- associated protein A (PAPP-A) (OR: 0.495, P = .023). The low expression of vascular endothelial growth factor (VEGF) (OR: 0.618, P = .005) was the influencing factor of embryo development arrest in early pregnancy. Building a prediction model based on the above indicators, it was found through testing that the random forest model is superior to the logistic regression model in predicting the risk of embryo development arrest. Conclusion • A random forest model based on age, ultrasound RI, progesterone, PAPP-A, ultrasound MSD/ CRL ratio, and VEGF index can help clinicians identify the risk of embryonic developmental arrest. [ABSTRACT FROM AUTHOR]

Context • Chromosomal abnormalities in embryos are the most common cause of early spontaneous abortions. Chromosome 1p36 deletion syndrome (OMIM 607872) is the most common subtelomeric, terminal microdeletion syndrome. Objective • The study intended to analyze miscarriage samples using chromosomal microarray analysis (CMA), to explore the mechanism of chromosomal aberrations, and to identify the recurrence risk and a prenatal diagnostic strategy for couples experiencing spontaneous abortions. Design • The research team performed a narrative review by searching PubMed databases. The search used the keywords 1p36 deletion, CMA, karyotype analysis, FISH and aborted fetus. The team also conducted case studies using genetic analyses. Setting • The study took place at Wuxi Maternity and Child Health Care Hospital in Wuxi, Jiangsu, PR China. Participants • Out of 673 abortion samples, six had 1p36 deletions (0.89%). Participants were the six families who had had those spontaneous abortions. Outcome Measures • The research team evaluated the fetal samples using: (1) CMA, (2) karyotype analysis, and (3) novel fluorescence in-situ hybridization (FISH). Results • The CMA showed that: (1) fetus 1 had a 1.75 MB microdeletion at the 1p36.32p36.31 band, which testing didn’t detect in fetus 1’s parents, but the research team couldn’t exclude the possibility that one of the parents was a carrier of a chromosomal insertional translocation; and (2) fetus 2 had a 5.10 MB microdeletion at the 1p36.13p36.12 segment, and fetus 3 had a 9.21 MB deletion at the 1p36.33p36.22 band, and the high-resolution karyotype analysis and FISH of the parents of both fetuses appeared normal, indicating that the chromosomal abnormalities were de novo; (3) fetus 4 had a 9.28 MB deletion at 1p36.33p36.22, although the high-resolution karyotype analysis of fetus 4’s parent was normal; (4) fetuses 5 and 6 had a 7.64 MB microdeletion at 1p36.33p36.23 and a 4.45 MB deletion at 1p36.33p36.32, respectively, although the parents of both fetuses waived further testing. Conclusions • This study provides the first report of recurrent spontaneous and sporadic abortions with 1p36 deletion syndrome. The CMA combined with a reasonable family-pedigree investigation can detect cryptic chromosomal aberrations in miscarriages and can determine the mechanism of the chromosomal variations. It thus is invaluable in assessing recurrence risk and providing appropriate prenatal diagnostic strategies for affected families. [ABSTRACT FROM AUTHOR]

In the last decade, the evolution of oncofertility has sparked a resurgence of interest in in vitro maturation (IVM) due to its suitability in certain oncological scenarios where controlled ovarian hyperstimulation may not be feasible. The retrieval of immature cumulus–oocyte complexes from small antral follicles, regardless of the menstrual cycle phase, presents a swift opportunity to vitrify mature oocytes or embryos post-IVM in urgent situations or when stimulation is not advisable. Harvesting immature cumulus–oocyte complexes and immature oocytes can be achieved transvaginally or directly in the laboratory from extracorporeal ovarian tissue. Although IVM has transitioned from an experimental status due to safety validations, it relies on the intricate process of oocyte maturation. Despite successful live births resulting from IVM in fertility preservation contexts, the comparatively lower developmental competence of in vitro matured oocytes highlights the necessity to enhance IVM culture systems. Recent advancements in IVM systems hold promise in bolstering oocyte competence post-IVM, thereby narrowing the gap between IVM and outcomes from ovarian stimulation. Additionally, for optimizing the chances of conception in cancer survivors, the combination of IVM and ovarian tissue cryopreservation stands as the favored choice when ovarian stimulation is unfeasible. [ABSTRACT FROM AUTHOR]

Poor ovarian response (POR) patients often face the risk of not having enough competent oocytes. Then, aspirating small follicles could serve as a strategy to increase their number. Many efforts have been addressed to associate follicular size with oocyte competence, but results are controversial. Therefore, our study aimed to evaluate oocyte maturation and developmental competence, along with a non-invasive oocyte-maturation-related miRNA signature in oocytes retrieved from both large and small follicles. A total of 178 follicles, from 31 POR patients, were aspirated and measured on the day of ovarian puncture. Follicular diameters, oocyte collection, oocyte maturation, fertilization, blastocysts, and good-quality blastocyst rates were recorded. Simultaneously, follicular fluids were collected to quantify their miRNA expression. The efficacy of oocyte retrieval along with oocyte maturation, fertilization, and blastulation rates tended to increase with follicular size, but few significant differences were found. Despite there being significantly more collected oocytes from follicles > 11.5 mm compared to follicles ≤ 11.5 mm (p < 0.05), oocytes from the latter were also mature, with no significant differences in the miRNA signature, but only those > 13.5 mm demonstrated developmental competence. In conclusion, 11.5 mm follicles can produce mature oocytes, but only those larger than 13.5 mm yielded transferable embryos. [ABSTRACT FROM AUTHOR]

This study aimed to compare the effectiveness, in terms of viability and quality post‐warming, when vitrifying in vitro‐produced (IVP) pig blastocysts with either a modified Cryotop (n = 161; 20 blastocysts/device) or the conventional Superfine Open Pulled Straw (SOPS; n=160; 5‐6 blastocysts/device systems. Blastocyst viability, morphology, and apoptosis parameters were evaluated after 24 h of in vitro culture. The Cryotop system yields better results in terms of higher embryo viability and total cell numbers (p <.05) and lower apoptosis (p <.05) parameters than the SOPS procedure, defining a high effectiveness to simultaneously vitrify 20 pig IVP blastocysts at one time, thus increasing the yield of IVP blastocysts readily available for embryo transfer. [ABSTRACT FROM AUTHOR]

The structural organization of eukaryotic genomes is contingent upon the fractionation of DNA into transcriptionally permissive euchromatin and repressive heterochromatin. However, we have a limited understanding of how these distinct states are first established during animal embryogenesis. Histone 3 lysine 9 trimethylation (H3K9me3) is critical to heterochromatin formation, and bulk establishment of this mark is thought to help drive large-scale remodeling of an initially naive chromatin state during animal embryogenesis. However, a detailed understanding of this process is lacking. Here, we leverage CUT&RUN to define the emerging H3K9me3 landscape of the zebrafish embryo with high sensitivity and temporal resolution. Despite the prevalence of DNA transposons in the zebrafish genome, we found that LTR transposons are preferentially targeted for embryonic H3K9me3 deposition, with different families exhibiting distinct establishment timelines. High signal-to-noise ratios afforded by CUT&RUN revealed new, emerging sites of low-amplitude H3K9me3 that initiated before the major wave of zygotic genome activation (ZGA). Early sites of establishment predominated at specific subsets of transposons and were particularly enriched for transposon sequences with maternal piRNAs and pericentromeric localization. Notably, the number of H3K9me3 enriched sites increased linearly across blastula development, while quantitative comparison revealed a >10-fold genome-wide increase in H3K9me3 signal at established sites over just 30 min at the onset of major ZGA. Continued maturation of the H3K9me3 landscape was observed beyond the initial wave of bulk establishment. [ABSTRACT FROM AUTHOR]

RNA-binding proteins (RBPs) play essential roles in coordinating germline gene expression and development in all organisms. Here, we report that loss of ADR-2, a member of the adenosine deaminase acting on RNA family of RBPs and the sole adenosine-to-inosine RNA-editing enzyme in Caenorhabditis elegans , can improve fertility in multiple genetic backgrounds. First, we show that loss of RNA editing by ADR-2 restores normal embryo production to subfertile animals that transgenically express a vitellogenin (yolk protein) fusion to green fluorescent protein. Using this phenotype, a high-throughput screen was designed to identify RBPs that when depleted yield synthetic phenotypes with loss of adr-2. The screen uncovered a genetic interaction between ADR-2 and SQD-1, a member of the heterogeneous nuclear ribonucleoprotein family of RBPs. Microscopy, reproductive assays, and high-throughput sequencing reveal that sqd-1 is essential for the onset of oogenesis and oogenic gene expression in young adult animals and that loss of adr-2 can counteract the effects of loss of sqd-1 on gene expression and rescue the switch from spermatogenesis to oogenesis. Together, these data demonstrate that ADR-2 can contribute to the suppression of fertility and suggest novel roles for both RNA editing–dependent and RNA editing–independent mechanisms in regulating embryogenesis. [ABSTRACT FROM AUTHOR]

We recorded the morphological characteristics and effect of preservation methods on the structure of the egg veils of Lophius litulon found in field investigations. The egg veils were characterized as translucent sheet‐shape with parallel opaque creases spaced approximately 2 cm apart. The egg veils were found to be composed of pentagonal or hexagonal chambers with rounded corners arranged in one layer, and each chamber enveloped one to three embryos. Cryopreservation is recommended to prevent structural changes in the egg veil rather than ethanol solution and neutral buffered formalin solution. [ABSTRACT FROM AUTHOR]

To study and compare the preimplantation genetic testing for monogenic disorders (PGT-M) results, and to evaluate the treatment cycle outcomes of embryos derived from a single pronucleus (1PN) vs. two pronuclei (2PN). A retrospective cohort study from January 2018 to December 2022 involving in vitro fertilization (IVF)-PGT-M treatment cycles. Single, academically affiliated fertility center. A total of 244 patients underwent 351 IVF-PGT-M treatment cycles. Embryo biopsy with molecular testing for a monogenic disorder. The molecular diagnosis results and clinical outcomes after the transfer of embryos derived from 1PN and 2PN in IVF-PGT-M treatment cycles. Embryos derived from 1PN have a significantly low developmental potential with a lower rate of embryos that underwent biopsy compared with 2PN-derived embryos; 1PN-derived embryos demonstrated a significantly lower number of blastocysts (24% vs. 37.9%) and top-quality blastocysts (22.3% vs. 48.1%) compared with 2PN-derived embryos. Lower successfully completed and unaffected PGT-M results were achieved in 1PN compared with 2PN-derived embryos (47.1% vs. 65.5% and 18.7% vs. 31.6%, respectively), with significantly higher abnormal molecular results (39.6% vs. 22.7%). The embryo transfer of 24 1PN-derived embryos with no affected genetic disorder resulted in 5 (20.8%) clinical pregnancies and 4 (16.7%) live births (LBs). Within the limits of fewer embryos derived from 1PN that yielded unaffected embryos suitable for transfer, the clinical pregnancy and LB rate of 1PN embryos undergoing PGT-M are reassuring. We, therefore, suggest applying PGT-M to embryos derived from 1PN embryos to improve the cumulative clinical pregnancy and LB rates. [ABSTRACT FROM AUTHOR]

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. Understanding how a sire influences early embryo survival, especially in dairy cattle, can enhance dairy industry efficiency and offer insights for other mammals. Investigating cellular stress in early embryos, this study found that low-performing sires, despite fertilising oocytes equally, may cause lower survival rates in the embryo's first week due to increased cellular stress mechanisms. Identifying this phenomenon sheds light on malfunctioning cellular mechanisms regulated by the sire, ultimately reducing embryo survival. Image by Lindsey Fallon. This article belongs to the Collection Dedication to Jim Cummins. [ABSTRACT FROM AUTHOR]

Somatic cell nuclear transfer (SCNT) is one of the primary methods for production of genetically engineered sheep, which allows for gene editing or transgene introduction in somatic cells. The use of SCNT eliminates the risk of genetic mosaicism in embryos and animals that is commonly observed after zygote micromanipulations. This retrospective analysis of SCNT in sheep performed at Utah State University, spanning from 2016 to 2021, examined parameters that may impact pregnancy and full-term development, including donor oocytes (donor age), donor cell lines, SCNT parameters (time of oocyte activation following SCNT, number of transferred embryos, in vitro maturation and culture conditions), and recipients (surgical number and ovulatory status), as well as factors that may correlate with large offspring syndrome or abnormal offspring syndrome (LOS/AOS) in the fetuses and lambs. Our findings indicated that compared to prepubertal oocytes, the SCNT embryos produced from adult sheep oocytes had comparable in vitro maturation rates, pregnancy and full-term development rates, as well as SCNT efficiency. In addition, earlier activation time of SCNT embryos (e.g. 24–26 h post maturation) was correlated to the early pregnancy loss rate, full-term rate, and SCNT efficiency. Compared to our standard serum-containing medium, commercial serum-free culture medium showed a positive correlation with the full-term development of sheep SCNT embryos. Transferring 15–30 embryos per recipient resulted in consistently good pregnancy rates. Surgical numbers and ovulatory status (having at least one follicle between 6 and 12 mm in size or a corpus hemorrhagicum (CH)) of recipients did not affect pregnancy and full-term development rates. In summary, this retrospective analysis identified parameters for improving pregnancy and full-term development of SCNT embryos in sheep. • The age of oocyte donors does not affect pregnancy rate of SCNT embryos, and SCNT efficiency. • Earlier activation time of SCNT embryos is correlated to the higher SCNT efficiency in sheep. • Surgical number and ovulatory status of recipients would not affect pregnancy and full-term development rates. [ABSTRACT FROM AUTHOR]

The effect of a fraction of phlorethol (CcPh), one of the polyphenolic compounds isolated from the brown alga Costaria costata (Turn.) Saund (Laminariales), on gametes and embryogenesis of the sea urchin Strongylocentrotus intermedius (A. Agassiz, 1864) was studied. Using a model of developing sea urchin embryos, it was shown that phlorethol at concentrations of ≤700 μg/mL did not exert a cytotoxic effect on early developmental stages (from the zygote to the 16-celled stage blastomere). The embryotoxic effect of phlorethol that caused the death of 50% of the embryos was expressed in a concentration (I) range of 50 < I ≤ 100 μg/mL only after 24 h of incubation, in the blastula stage. When exposed to concentrations greater than 100 μg/mL, embryos that survived to the blastula stage did not develop further and died after 36 h of incubation. Under the effect of phlorethol, the fertilizing capacity of sea urchin spermatozoa and oocytes decreased. The concentration of phlorethol that inhibited fertilization by 50% (IC50) when acting on sperm was 1.28 ± 0.38 μg/mL and when acting on oocytes was 3.83 ± 0.82 μg/mL. Thus, phlorethol reduces the fertilizing capacity of sea urchin gametes at concentrations much lower than embryotoxic concentrations and can potentially find practical application as part of new contraceptive drugs for mammals and humans. [ABSTRACT FROM AUTHOR]

After normal pollination and fertilization of pseudoparthenocarpic seedless grapes, their embryos often stop developing due to certain developmental factors, resulting in embryo abortion. Hybrid breeding using seedless grapes as the maternal parent requires embryo rescue breeding technology. This technology plays a crucial role in seedless grape breeding. Although previous studies have extensively explored this technology, knowledge regarding its impact on embryo abortion and the effectiveness of rescue techniques remains limited. This study aimed to investigate the correlation between embryo rescue and hormonal changes in seedless grapes. Four Eurasian seedless grape cultivars, "Thompson Seedless," "Flame Seedless," "Heshi Seedless," and "Ruby Seedless," were selected for examination. We investigated endogenous hormone levels, including indole-3-acetic acid (IAA), gibberellic acid (GA3), and abscisic acid (ABA), in both berries and in vitro ovules during the most suitable embryo rescue time for these cultivars. Based on the observed fluctuations in endogenous hormone levels and previous research findings, appropriate doses of exogenous hormones, such as IAA, GA3, and ABA, were applied during seedless grape embryo rescue. The results indicated significant differences in endogenous hormone levels between berrieswith varying ovule counts of the same cultivar and ovules cultured in vitro, suggesting a hormonal influence on ovule abortion and embryo development in seedless grapes. Further research has identified effective ratios of exogenous hormones: 30 mg·L-1 IAA + 30 mg·L-1 ABA for berry ovule development, 1.0 mg·L-1 IAA + 2.0 mg·L-1 6-benzylaminopurine (6-BA) + 1.0 mg·L-1 GA3 + 1.0 mg·L-1 ABA for in vitro ovule development, and 1.0 mg·L-1 IAA + 2.0 mg·L-1 6-BA + 1.0 mg·L-1 GA3 for embryo germination and seedling formation. In summary, hormonal changes significantly influence ovule and embryo development and are closely related to seedless grape embryo rescue breeding. This study deepened our understanding of the correlation between seedless grape embryo rescue and hormonal changes. It also resulted in the successful production of a batch of embryo rescue seedlings, further improving embryo rescue breeding technology and providing new germplasm materials for developing new seedless grape cultivars. [ABSTRACT FROM AUTHOR]

The article investigates a little‐known work titled De ponderibus et mensuris [On Weights and Measures] written by Juan de Mariana in 1599. It contains the embryo of his future monetary theories, and has other interesting aspects, not only juridical and historical but also epistemological. Significant points of convergence with the early modern mathematics and abacus tradition make it unique. Apart from its stylistic, pedagogical and technical originality, this work also appears as a watershed for the evolution of economic science, its language and methodology. [ABSTRACT FROM AUTHOR]

We studied the development of sporocysts of the trematode Leucochloridium paradoxum. For this purpose, their life cycle was traced experimentally. Eggs obtained from adult trematodes raised in chicks (Gallus gallus domesticus) were used to infect snails (Succinea putris, laboratory strain). Histological studies of sporocysts were conducted for 95 days after exposure. During this time, the embryos of sporocysts developed into a small stolon filled with numerous metacercarial embryos. We found that miracidia hatched in the stomach and in the proximal part of the snail's midgut. After the larvae migrated into the area of the hepatopancreas, their body was destroyed, and germinal cells were released. They cleaved to form the embryos of sporocysts, in which only metacercarial embryos developed. Our study showed that these trematodes lack the parasitic phase of the mother sporocyst and have only one generation of parthenitae, the daughter sporocysts. [ABSTRACT FROM AUTHOR]

Pre-implantation genetic testing for aneuploidy (PGT-A) is used in approximately half of in vitro fertilization cycles. Given the limited understanding of the genetics of human embryos, the current use of PGT-A is based on biologically uncertain assumptions and unvalidated guidelines, leading to the possibility of disposing of embryos with pregnancy potential. We isolated and sequenced all single cells (1133) from in vitro cultured 20 human blastocysts. We found that all blastocysts exhibited mosaicism with mitotic-induced aneuploid cells and showed an ~25% aneuploidy rate per embryo. Moreover, 70% (14/20) of blastocysts contained 'chromosome-complementary' cells, suggesting genetic mosaicism is underestimated in routine PGT-A. Additionally, the analysis of 20,945 single cells from day 8–14 embryos (in vitro cultured) and embryonic/fetal organs showed that 97% of the analyzed embryos/organs were mosaic. Over 96% of their aneuploid cells harbored ≤ 2 chromosome errors. Our findings have revealed a high prevalence of mosaicism in human embryos. [ABSTRACT FROM AUTHOR]

Simple Summary: The combination of JAK/HDAC inhibitors shows beneficial effects in hematological malignancies and a promising therapeutic potential in CTCL. Recent in vitro and in vivo data have shown that the combination of Resminostat (HDACi) with Ruxolitinib (Jaki) acts synergistically and inhibits tumorigenesis and metastasis in CTCL experimental models. Angiogenesis is the most critical process for tumor growth and metastasis and plays an essential role in cutaneous lymphoma development and progression. The vascular microenvironment of lymphomas accelerates angiogenesis via the paracrine activity of tumor-derived mediators. However, few studies have investigated the role of either ruxolitinib or resminostat in the angiogenesis of hematological malignancies and solid tumors. Although precise mechanisms of angiogenesis in CTCL remain unclear, the chick embryo chorioallantoic membrane (CAM) has been used, among other in vivo models, to implant several tumor types as well as malignant cell lines to study their growth rate, angiogenic potential, and metastatic capability. The aim of this study was to investigate the effects of combinational therapies of JAKi/HDACi in CTCL angiogenesis by using a CTCL-specific chick embryo model. Angiogenesis plays a pivotal role in the growth and metastasis of tumors, including the development and progression of cutaneous lymphomas. The chick embryo CAM model has been utilized in various studies to assess the growth rate, angiogenic potential, and metastatic capability of different tumor types and malignant cell lines. However, the precise mechanisms of angiogenesis in CTCL and the influence of Ruxolitinib or Resminostat on angiogenesis in hematological malignancies and solid tumors are not well understood. Recent in vitro and in vivo data have demonstrated the synergistic inhibition of tumorigenesis and metastasis in experimental models of CTCL when using the combination of Resminostat (HDACi) with Ruxolitinib (JAKi). The present work aims to elucidate the effects of this combination on the tumor microenvironment's vascular components. We investigated the effects of Ruxolitinib (JAKi) in combination with Resminostat (HDACi) treatment in transendothelial migration of CTCL cells (106 MyLa and SeAx) by using a transwell-based transendothelial migration assay and tumor angiogenesis in vivo. We used the CTCL chick embryo CAM model with xenografted tumors derived from implanted MyLa and SeAx cells and administered topically 15 μM ruxolitinib and 5 μM Resminostat every two days during a 5-day period. JAKi and HDACi inhibited CTCL cell transendothelial migration by 75% and 82% (p < 0.05) in both CTCL engrafted cells (MyLa and SeAx, respectively) compared to the untreated group. Moreover, the combination of ruxolitinib with resminostat blocked angiogenesis by significantly reducing the number of blood vessel formation by 49% and 34% in both MyLa and SeAx, respectively (p < 0.05), indicating that the proposed combination exerted significant anti-angiogenic effects in the CAM CTCL model. Overall, these data provide valuable insights into potential therapeutic strategies targeting angiogenesis in CTCL, paving the way for more effective treatment approaches in the future. [ABSTRACT FROM AUTHOR]

Integrating in vitro embryo production with embryonic microsurgery facilitates the generation of monozygotic twins. However, despite their potential benefits, these methods have not been widely adopted in commercial settings because of their substantial costs. Hence, there is a need to streamline the bisection procedure while ensuring efficient production of viable demi-embryos. In this study, we investigated the impact of different orientations of microsurgical incisions in relation to inner cell mass on embryonic development, morphology, viability, and expression of cell fate protein markers using a simplified microsurgery approach. Ovaries were transported from the slaughterhouse to the laboratory and aspirated to obtain oocytes that were selected and subjected to in vitro embryo production. The selected expanded blastocysts (n = 204) underwent microsurgery. The blastocysts were immobilized to facilitate incision using an adapted microblade, yielding demi-embryos (vertical incision) and viable embryonic fragments (transverse incision). The structures were then re-cultured for 12 h. Viability was assessed by measuring the re-expansion rate after re-culture, followed by immunofluorescence analysis of proteins (CDX2 and NANOG) and apoptosis analysis using terminal deoxynucleotyl transferase dUTP nick end-labeling (TUNEL). Microsurgically derived embryos exhibited remarkable plasticity, as evidenced by a slight reduction (P < 0.05) in the re-expansion rate (transverse 64.2 % and vertical 57.2 %) compared to that of the control group (blastocysts without microsurgery) (86.7 %). They also demonstrated the ability of morphological reconstitution after culturing. Despite the anticipated decrease (P < 0.05) in the total number of cells and embryo volume, microsurgery did not result in a significant increase (P > 0.05) in the number of apoptotic cells. Furthermore, microsurgery led to higher (P < 0.05) expression of markers associated with pluripotency, indicating its efficiency in preserving regenerative capacity. Moreover, microsurgery, whether followed by immunosurgery or not, made the isolation of embryonic cells easier. In conclusion, both transverse and vertical microsurgery incisions enabled the production of identical demi-embryos and served as tools for isolating embryonic cells without compromising the resumption of development and the apoptotic index. • Different microsurgical incision orientations on embryonic viability. • Cellular and molecular blastocyst reconstitution characterization. • Re-expansion rates after microsurgery demonstrate incredible embryonic plasticity. • Embryonic microsurgery is a tool for obtaining demi-embryos and embryonic cells. [ABSTRACT FROM AUTHOR]

The aim of this study was to evaluate the superestimulatory and superovulatory responses of cattle treated with corifollitropin-alpha, a long-acting human recombinant FSH (rhFSH). In the first and second experiments, we used Nelore (Bos indicus) heifers previously submitted to follicular wave suppression by active immunization against GnRH. In Experiment 1 (a dose-response study), heifers (n = 20) were randomly allocated into five groups, which received placebo (saline) or a single sc dose of 7.5, 15.0, 22.5 or 30.0 μg rhFSH. The heifers were subjected to daily ovarian scan and blood sampling during 11 days. We observed group, time, and group x time effects (P< 0.0001) for both average follicle size and circulating FSH concentrations, with a strong correlation (R = 0.82, P< 0.0001) between the area under curve (AUC) for both parameters. The peak concentration of FSH 24h after treatment and average follicle size at all timepoints, however, were similar (P> 0.05) between groups 22.5 and 30.0 μg. In Experiment 2, heifers (n = 18) were allocated into three groups, which received (0h) either placebo (control), 25 μg rhFSH or 130 mg pFSH (Folltropin). There was no difference (P> 0.05) in average follicle size at any moment, as well as in intrafollicular E2 at 120h or in plasma P4 seven days later between groups rhFSH and pFSH. In Experiment 3, cycling Nelore heifers (n = 20) were subjected to a wave synchronization protocol and superovulated (day 0) using a standard pFSH protocol (120 mg split in eight decreasing im doses) or with a single sc injection of 20 μg rhFSH. The number of follicles >7 mm on day 4 did not differ (P= 0.4370). Heifers receiving rhFSH had greater average follicle size on day 4 (P= 0.0005), ovulation rate (P< 0.0001), and number of CL (P= 0.0155), as well as a trend towards a greater number of ova (P= 0.07) and viable embryos (P= 0.0590). In Experiment 4, superovulation was induced with a single sc injection of 25 μg rhFSH in Girolando and Nelore cows and heifers (n = 20). None of the embryo yield endpoints differed between the two breeds (P> 0.05). In conclusion, cattle superstimulation and superovulation can be successfully induced with a single dose of a long-acting rhFSH (corifollitropin-alpha). • Cattle respond to superstimulation with corifollitropin-alpha in a dose-dependent manner. • Ovarian superovulation can be induced with 20–25 mcg of corifollitropin-alpha. • Corifollitropin-alpha is detected in circulating serum up to 11 days after treatment. [ABSTRACT FROM AUTHOR]

METTL3-mediated N6-methyladenosine (m6A) modification is critical for gametogenesis and early embryonic development. However, the function of METTL3-mediated m6A modification in the early development of somatic nuclear transfer embryos (SCNT) remains unclear. Here, we found that METTL3 mRNA and protein levels exhibit dynamic changes during the early development of porcine SCNT embryos. The levels of METTL3 mRNA and protein in SCNT embryos at specific developmental stages differ from those in parthenogenetic activation (PA) counterparts. SiRNA injection effectively reduced the levels of METTL3 mRNA and protein in 4-cell embryos and blastocysts. METTL3 knockdown significantly reduced the cleavage and blastocyst rates of SCNT embryos. METTL3 knockdown significantly reduced the number of total cells and trophectoderm (TE) cells in the resulting blastocysts and perturbed cell lineage allocation. In addition, METTL3 knockdown reduced the levels of m6A modification in 4-cell embryos and blastocysts. Importantly, METTL3 knockdown decreased the expression levels of CDX2 , GATA3 , NANOG and YAP , and increased the expression levels of SOX2 and OCT4. Taken together, these results demonstrate that METTL3-mediated m6A modification regulates early development and lineage differentiation of porcine SCNT embryos. [ABSTRACT FROM AUTHOR]

This work explored whether a well-characterized recombinant human interleukin-6 (hIL6) protein will influence in vitro produced (IVP) bovine embryo development and survival after cryopreservation. Cumulus oocyte complexes were collected from abattoir derived ovaries, matured for 24 h, and fertilized using pooled semen from Holstein bulls. Embryos were treated with 0, 25, 50, or 100 ng/mL hIL6 on day 5 post-fertilization. An increase in ICM cell numbers was observed in each hIL6 treatment, with the lowest hIL6 treatment having the same magnitude of response as the middle and highest hIL6 concentration. No effects on TE cell numbers were observed. The second study involved cryopreserving (via slow freezing) of hIL6-treated blastocysts, then examining post-thaw blastocyst survival by incubating for 24 h in the absence of hIL6 treatments. Blastocyst re-expansion and hatching rates were unaffected by any of the IL6 treatments, however, increases in both ICM and TE cell numbers were detected at 24 h post-thawing in blastocysts exposed to 100 ng/mL hIL6 but not lower concentrations before freezing. A reduction in the percentage of TUNEL-positive TE cells was observed after thawing in blastocysts exposed to 25, 50 and 100 ng/mL hIL6 before cryopreservation. No treatment-dependent changes in TUNEL-positive ICM cells were observed. In summary, hIL6 supplementation improves ICM cell numbers in bovine blastocysts to a degree that is commensurate with what has been observed when using bovine recombinant IL6. This positive effect of hIL6 on ICM cell numbers is maintained after freezing and thawing, and a novel improvement in post-thaw TE cell numbers occur in hIL6 treated embryos. This positive effect on TE cell numbers is attributed, at least in part, to an hIL6-dependent reduction in TE cell apoptosis. [Display omitted] • Increases in inner cell mass cell numbers are achieved by supplementing recombinant human interleukin-6. • Positive effects of human interleukin-6 supplementation on both the inner cell mass and trophectoderm were detected after embryo cryopreservation. • Human interleukin-6 may is effective in bovine embryos. [ABSTRACT FROM AUTHOR]

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. • The presence of oviductal epithelial cells during embryo in vitro culture enhanced porcine blastocyst formation. • The oviductal epithelial cells during embryo culture decrease ROS levels in porcine in vitro produced cleaved embryos. • The porcine oviductal epithelial cells expressed OVGP-1 during in vitro culture. [ABSTRACT FROM AUTHOR]

This study examined how the vitrification of pig blastocysts using either the superfine open pulled straw (SOPS) or Cryotop method affects the expression profile of embryonic microRNA (miRNA) transcriptomes, as well as its relation to changes in the expression of target genes (TGs). Surgically collected pig blastocysts were vitrified using either the SOPS method (n = 60; 4–6 embryos/device) or the Cryotop system (n = 60; 20 embryos/device). Embryos were cultured in vitro for 24 h after warming. Fresh blastocysts (n = 60) cultured for 24 h served as controls. After in vitro culture, five pools of eight viable blastocysts from each group were prepared for miRNA expression analysis based on a microarray approach. Then, biological interpretation of miRNAs profiles and integrative analysis of miRNA and mRNA transcriptome data were performed. Survival after 24 h of in vitro culture was similar (>96 %) for both the vitrification systems and the control group (100 %). Compared with the controls, the SOPS-vitrified blastocysts had 94 (one upregulated and 93 downregulated) differentially expressed (DE) miRNAs, and the Cryotop-vitrified blastocysts had 174 DE miRNAs (one upregulated and 173 downregulated). One DE miRNA (miR-503) in the SOPS group and three DE miRNAs (miR-7139–3p, miR-214 and miR-885–3p) in the Cryotop group were annotated for Sus scrofa. The integrative analysis showed that 27 and 61 DE TGs were regulated by the DE miRNAs in blastocysts vitrified with the SOPS and Cryotop systems, respectively. The TGs enriched one pathway (the TGF-β signaling pathway) for the SOPS system and four pathways (HIF-1, Notch, ascorbate and aldarate metabolism and glycosphingolipid biosynthesis-ganglio series) for the Cryotop system. In summary, vitrification via the SOPS and Cryotop systems dysregulates miRNAs, with slight differences between methods. The altered miRNAs identified in this study were related mainly to cell proliferation, apoptosis, and the response to cell stress. Further studies are needed to clarify the consequences of dysregulation of miRNAs involved in the TGF-β (SOPS-vitrified blastocyst) and Notch (Cryotop-vitrified blastocyst) signaling pathways, particularly if they can affect embryonic development. • Vitrification modifies miRNA expression in porcine blastocysts. • SOPS and Cryotop-vitrified embryos showed minor differences in miRNA expression. • miRNAs target genes were related to proliferation, apoptosis, and stress response. • The modified miRNAs had a regulatory function in response to vitrification. [ABSTRACT FROM AUTHOR]

The present investigation was aimed at predicting a still (i.e., dead) vs. live embryo within a hatching goose egg by measuring the eggshell cooling rate. For this, we daily measured the temperature (T) values on the shell surface of goose eggs after they were removed from the incubator and during further natural cooling. T was recorded every 0.5 h for further 1.5 h of cooling. It was possible to recognize eggs with dead embryos using the combination of T , egg weight (W), and surface area (S). The resultant indicator (TS / W) was called specific temperature index (STI). The mathematical relationship using STI measurements between Days 8–13 facilitated 80 % correct identification of the eggs with dead embryos. Additionally, we derived mathematical dependencies for shell weight (W s) and thickness (t) by utilizing the values of W , egg volume (V), S , the average T of all measurements taken, as well as the drop in T during 1.5 h of natural cooling. The key advantage of these parameters was their measurement and/or calculation by applying non-destructive methods. The integrated application of these parameters resulted in achieving high calculation accuracy as judged by correlation coefficients of 0.908 for W s and 0.593 for t. These novel mathematical models have the potential to decrease hatching waste by predicting embryo viability. Our research will add to a toolkit for non-invasive egg assessment that is useful in the poultry industry, research on eggs, and engineering. [Display omitted] • We aimed at predicting still (dead) vs. live embryos in goose eggs by cooling rate. • We measured shell surface temperature (T) during natural cooling for 1.5 h. • Specific temperature index was derived to predict correct identification of eggs. • We propose this index to predict eggs with dead embryos and reduce hatching waste. [ABSTRACT FROM AUTHOR]

Previous studies indicate a positive correlation between the duration of estrus prior to ovulation and likelihood of pregnancy in embryo recipient mares. However, the mechanisms by which the duration of estrus before may affect fertility remains unclear. This study aimed to determine the effect of different durations of estradiol exposure, prior to progesterone administration, on embryo viability in anestrous recipient mares, and endometrial expression of genes thought to influence embryo survival. Three groups of anestrous recipient mares treated with different duration of estradiol were used: long (LE), short (SE) and no treatment (NE). Day 8 embryos were transferred into recipient mares four days after long-acting progesterone administration and recovered 48h later to examine embryo growth and viability. The endometrial gene expression profile of selected genes was also investigated. The likelihood of recovering an embryo 48h after transfer was 46.1% (6/13), 62.5% (5/8) and 85.7% (6/7) for recipient mares from the NE, SE and LE groups, respectively (P =.09). Embryos recovered from the different groups of recipients did not, however, differ in size, morphology or the proportion of nuclei undergoing mitosis (P >.05). Abundance of mRNA for uterocalin (P19) and insulin-like growth factor 1 (IGF1) were increased in the LE compared to the NE group, while fibroblast growth factor 2 (FGF2), progesterone receptor (PGR) and insulin-like growth factor 1 receptor (IGF1R) transcript abundances were increased (P < 0.05) in the NE group compared to both SE and LE groups. In conclusion, a longer exposure of the endometrium to estradiol before progesterone tended to improve embryo survival within 48h of transfer. However, the grade, growth rate, and proportion of mitotic cells in surviving embryos did not differ among groups. If embryos are destined to fail in a suboptimal endometrial environment, they die and disappear quickly. Moreover, a more adequately estradiol-primed uterus, before the progesterone rise, seems to create a uterine environment, in terms of P19 , IGF1 , FGF2 and PGR gene expression, more conducive to embryo survival and further development. • Embryo survival after transfer tended to be influenced by the duration of estradiol treatment. • Viability of surviving embryos within 48h of transfer was not affected by estradiol-primed uterus. • Endometrial expression of selected genes was influenced by estradiol-primed uterus. [ABSTRACT FROM AUTHOR]

Background: MicroRNAs (miRNAs) are presented in the uterine lumen of many mammals, and in vitro experiments have determined that several miRNAs are important for the regulation of endometrial and trophoblast functions. Our aim was to identify and contrast the miRNAs present in extracellular vesicles (EVs) in the uterine lumen fluid (ULF) at the onset of attachment in cattle pregnancies (gestation d 18) initiated by artificial insemination (AI) or by the transfer of an in vitro-produced blastocyst (IVP-ET). A third group had no conceptus after the transfer of an IVP embryo. Results: The abundance of 263 annotated miRNAs was quantified in the EVs collected from ULF. There was an increase in the transcript abundance of 20 miRNAs in the ULF EVs from the AI pregnant group, while 4 miRNAs had a lower abundance relative to the group not containing a conceptus. Additionally, 4 miRNAs were more abundant in ULF EVs in the AI pregnant group relative to IVP-ET group (bta-mir-17, bta-mir-7-3, MIR7-1, MIR18A). Specific miRNAs in the ULF EVs were co-expressed with messenger RNAs expressed in extra-embryonic tissues and endometrium, including genes that are known to be their targets. Conclusions: The results provide biological insights into the participation of miRNAs in the regulation of trophoblast proliferation and differentiation, as well as in endometrium receptivity. The knowledge that in vitro cultured embryos can contribute to the altered abundance of specific miRNAs in the uterine lumen can lead to the development of corrective approaches to reduce conceptus losses during the first month of pregnancy in cattle. [ABSTRACT FROM AUTHOR]