Stages Of Embryo Development Research Articles

Speed breeding advancements in safflower (Carthamus tinctorius L.): a simplified and efficient approach for accelerating breeding programs.

This study investigated the potential of extended irradiation combined with immature embryo culture techniques to accelerate generation advancements in safflower (Carthamus tinctorius L.) breeding programs. We developed an efficient speed breeding method by applying light-emitting diodes (LEDs) that emit specific wavelengths, alongside the in vitro germination of immature embryos under controlled environmental conditions. The experimental design for light treatments followed a 2 × 4 completely randomized factorial design with four replications, incorporating two safflower varieties, Remzibey-05 and Dinçer, and four LED treatments (white, full-spectrum, red + blue + white, and control). A lighting regimen of 22 h of light and 2 h of darkness was applied for all the LED treatments, whereas the control received 18 h of light and 6 h of darkness. Additionally, the immature embryo culture experiment used a 2 × 2 × 4 factorial arrangement, assessing two safflower cultivars, two media types, and four embryo developmental stages, with three replications. The parameters evaluated included plant height, branch number, seed number per plant, seed number per head, time to flower initiation, time to 50% flowering, time to harvest, and germination percentage of in vitro cultured immature embryos at various developmental stages. The harvest time among the light treatments ranged from 50.62 to 73.12days, with the shortest time achieved under the red + blue + white LED combination and the longest under the control treatment. The plant height, number of seeds per plant, and number of seeds per head were highest under the full-spectrum LED, control and red + blue + white LED combinations, respectively. Immature embryos rescued at 10dayspost-pollination presented a 57% germination rate, with an increasing trend in germination as the number of dayspost-pollination increased. The germination rates did not significantly differ across varieties or hormone treatments. This study demonstrated the potential to achieve six generations per year by combining prolonged illumination with targeted LED lighting and immature embryo culture techniques. These findings provide valuable insights for optimizing safflower growth and development and advancing speed breeding in controlled environments.

Integrative Protocol for Generation of iPSC-Derived 3D Human Hepatic Organoids.

Obtaining stable hepatic cells in culture poses a significant challenge for liver studies. Bearing this in mind, an optimized method is depicted utilizing human induced pluripotent stem cells (hiPSCs) to generate 3D cultures of human hepatic organoids (HHOs). The utilization of HHOs offers a valuable approach to understanding liver development, unraveling liver diseases, conducting high-throughput studies for drug development, and exploring the potential for liver transplantation. In the former investigation, through immunofluorescence and quantitative RT-PCR techniques, the progression was monitored, identifying the presence of various cell populations, such as hepatoblasts and the two types of hepatoblast-derived cells: cholangiocytes or hepatocyte-like cells, across different developmental stages. This report presents a straightforward 3D protocol starting from hiPSC to acquire HHOs that mirror the stages of human embryo development. The protocol, spanning 46-50 days, encompasses several steps: (i) meticulous management of hiPSC culture to generate HHOs, (ii) initiation of cell differentiation in 2D and the subsequent transition to 3D, and (iii) an optimized dissociation strategy to break down HHOs into single cells for single-cell RNA sequencing. As an illustration of the broad applications of this approach, the present protocol was previously applied to unravel the role of thyroid hormone signaling in developing liver cells.

LINE-1-Induced Retrotransposition Affects Early Preimplantation Embryo DNA Integrity and Pluripotency.

Retrotransposable elements are implicated in genome rearrangements and gene expression alterations that result in various human disorders. In the current study, we sought to investigate the potential effects of long interspersed elements-1 (LINE-1) overexpression on the integrity and methylation of DNA and on the expression of three major pluripotency factors (OCT4, SOX2, NANOG) during the preimplantation stages of human embryo development. Human MI oocytes were matured in vitro to MII and transfected through intracytoplasmic sperm injection (ICSI) either with an EGFP vector carrying a cloned active human LINE-1 retroelement or with the same EGFP vector without insert as control. The occurrence of retrotransposition events was screened by fluorescent microscopy. The in vitro preimplantation development as well as the methylation, pluripotency, and DNA double-strand breaks (DSBs) of the transfected embryos were examined. LINE-1 retrotransposons gave rise to new retrotransposition events in the transfected embryos. LINE-1 injected embryos were characterized by accelerated asymmetrical cell division, multiple cellular fragments, cleavage arrest, and degeneration. Early OCT4 expression remained unaltered, but cleavage arrest and a high fragmentation rate hindered the expression of SOX2/NANOG at the morula stage. Increased DNA DSBs were observed in cleavage-stage blastomeres, while no methylation changes were detected before the cleavage arrest. Our data provide evidence that LINE-1 retrotransposition in human preimplantation embryos may induce DNA DSBs, while at the same time, it appears to interfere with the expression patterns of pluripotency factors. The morphological, structural, and cleavage abnormalities of the transfected embryos show that aberrant retroelement expression may negatively affect human embryo development.

A Combined Fertility and Developmental Toxicity Study with an Antisense Oligonucleotide Targeting Murine Apolipoprotein C-III mRNA in Mice.

Here, we present the reproductive toxicology profile of ISIS 838707, a GalNAc-conjugated antisense oligonucleotide (ASO) targeting mouse Apolipoprotein C-III (ApoC-III) mRNA. ISIS 838707 was subcutaneously administered during the premating, mating, and gestation periods to male and female mice at 0, 5, 10, and 20 mg/kg/week. Key focus areas included fertility, reproductive cell functions, estrus cycle, tubal transport, implantation, embryo development stages, and teratogenic potential. We also investigated the toxicokinetics and target mRNA knockdown effects. The treatment was well-tolerated at all dose levels, with no overt toxicity. Treatment led to decreased total cholesterol and/or triglyceride levels at doses ≥5 mg/kg/week, concordant with effective knockdown of ApoC-III mRNA (>85% reduction at all dose levels). Toxicokinetic analysis revealed predominant distribution to the liver of parental animals and minimally to the placenta, with no detectable transfer to fetal liver. Despite these pharmacological effects, there were no discernible adverse impacts on developmental and reproductive functions. These findings suggest that ISIS 838707, while effective in modulating ApoC-III mRNA and lipid profiles, does not adversely impact on reproductive and developmental functions in mice. The study contributes insights into the safety profile of ASOs and reduction of ApoC-III expression, particularly in the context of reproductive and developmental health.

The composition of commercially available human embryo culture media.

What is the composition of currently available commercial human embryo culture media provided by seven suppliers, for each stage of human preimplantation embryo development? While common trends existed across brands, distinct differences in composition underlined the absence of a clear standard for human embryo culture medium formulation. The reluctance of manufacturers to fully disclose the composition of their human embryo culture media generates uncertainty regarding the culture conditions that are used for human preimplantation embryo culture. The critical role of the embryo culture environment is well-recognized, with proven effects on IVF success rates and child outcomes, such as birth weight. The lack of comprehensive composition details restricts research efforts crucial for enhancing our understanding of its impacts on these outcomes. The ongoing demand for greater transparency remains unmet, highlighting a significant barrier in embryo culture medium optimization. For this study, 47 different human embryo culture media and protein supplements were purchased between December 2019 and June 2020; they comprise complete media (n = 23), unsupplemented media (n = 14), and supplements (n = 10). Unsupplemented media were supplemented with each available supplement from the same brand (n = 33 combinations). All samples were directly frozen in liquid nitrogen and stored at -80°C until composition analysis. We determined the concentrations of 40 components in all samples collected (n = 80). Seven electrolytes (calcium, chloride, iron, magnesium, phosphate, potassium, sodium), glucose, immunoglobulins A, G, and M (IgA, IgG, IgM), uric acid, alanine aminotransferase (ALAT), aspartate aminotransferase (ASAT), and albumin, as well as the total protein concentration, were determined in each sample using a Cobas 8000 Analyser (Roche Diagnostics). Analysis of pyruvate, lactate, carnitine, and 21 amino acids was achieved with Ultra-High Performance Liquid Chromatography-Mass Spectrometry (UPLC-MS/MS). Our analysis showed that generally, the concentrations of components of ready-to-use human embryo culture media align with established assumptions about the changing needs of an embryo during early development. For instance, glucose concentrations displayed a high-low-high pattern in sequential media systems from all brands: 2.5-3 mM in most fertilization media, 0.5 mM or below in all cleavage stage media, and 2.5-3.3 mM in most blastocyst stage media. Continuous media generally resembled glucose concentrations of cleavage stage media. However, for other components, such as lactate, glycine, and potassium, we observed clear differences in medium composition across different brands. No two embryo culture media compositions were the same. Remarkably, even embryo culture media from brands that belong to the same parent company differed in composition. Additionally, the scientific backing for the specific concentrations used and the differences in the composition of sequential media is quite limited and often based on minimal in vivo studies of limited sample size or studies using animal models. N/A. We used a targeted approach and performed a selection of tests which limit the composition analysis to this set of analytes. Comprehensive disclosure and complete transparency concerning the composition of human embryo culture media, including the exact concentration of each component, are crucial for evidence-based improvements of culture media for human preimplantation embryos. This research was supported by ZonMw (https://www.zonmw.nl/en), Programme Translational Research 2 (project number 446002003). M.G. declares an unrestricted research grant from Ferring not related to the presented work, paid to the institution VU Medical Center. The remaining authors have no conflicts of interest to declare. N/A.

Spatiotemporal characterization of cyclooxygenase pathway enzymes during vertebrate embryonic development

Vertebrate development is regulated by several complex well-characterized morphogen signaling pathways, transcription factors, and structural proteins, but less is known about the enzymatic pathways that regulate early development. We have identified that factors from the inflammation-mediating cyclooxygenase (COX) signaling pathway are expressed at early stages of development in avian embryos. Using Gallus gallus (chicken) as a research model, we characterized the spatiotemporal expression of a subset of genes and proteins in the COX pathway during early neural development stages. Specifically, here we show expression patterns of COX-1, COX-2, and microsomal prostaglandin E synthase-2 (mPGES-2) as well as the genes encoding these enzymes (PTGS1, PTGS2, and PTGES-2). Unique expression patterns of individual players within the COX pathway suggest that they may play non-canonical/non-traditional roles in the embryo compared to their roles in the adult. Future work should examine the function of the COX pathway in tissue specification and morphogenesis and determine if these expression patterns are conserved across species.

A kind of protein affect the expression of bnk

The maternal gene products control the initial stages of embryo development in all animals. The role of controlling embryogenesis is taken up by the zygotic genome at some point after fertilization. This paper seeks to shed light on research on the effect of the zinc-finger protein Zelda on the expression of bnk in fruit flies. The zinc-finger protein Zelda (Zld) is a transcriptional activator of the early zygotic genome and plays an important role in early embryonic development. (Liang,2008) This paper mainly demonstrates the influence of Zld on the bnk, which encodes a kind of protein that controls the actinic organization and the time of cellular basal closure. In the absence of Zelda, the expression of bnk has subtle changes. This work highlights the importance of the zinc-finger protein Zelda in the expression of the bnk in fruit flies and also details the methodology followed in the research on the bnk expression in fruit flies, the results, and the conclusions drawn from the research work, showing the importance of Zld to bnk, especially in early embryonic development stages. The significance of this research is to open potential avenues for research on identifying genes whose expression has been influenced to some extent by Zelda. Also, understanding the effect of Zelda on bnk expression can aid in the manipulation of the two variables to establish a functional interaction.

Assessment of loliolide extracted from Biserula pelecinus, present during in vitro oocyte maturation, on fertilisation and embryo development in sheep

Context As a ‘duty of care’, it is important to test whether new forage plants for ruminants contain secondary compounds (PSCs) that affect reproductive performance. We have previously observed, a posteriori, that the presence of a methanolic extract of Biserrula pelecinus during maturation of sheep oocytes increased fertilisation rate and blastocyst development. This result needed to be verified a priori and, if the outcome was repeated, we needed to identify the plant secondary metabolite responsible. Aims To test whether PSCs from B. pelecinus, when added to the oocyte maturation medium, improve fertilisation rate and blastocyst development; to test whether loliolide is the active molecule produced by B. pelecinus. Methods Methanol–chloroform extracts of B. pelecinus were fractionated using rapid silica filtration and solvents of increasing polarity. Fractions at final concentrations of 0, 100 or 200 μg mL−1 were added to the medium used to mature sheep cumulus–oocyte complexes (COCs) and effects were determined for maturation, subsequent cleavage rate, blastocyst rate, hatching rate, blastocyst efficiency and total blastocyst cell number (TCN). Results Fraction BP-6 at 100 μg mL−1 reduced blastocyst rate (P < 0.05), but had no effect when the dose was doubled to 200 μg mL−1. Further fractionation using semi-preparative high-performance liquid chromatography showed loliolide as the most abundant compound in BP-6. Supplementation of the in vitro maturation medium with loliolide (0, 2.5, 5, 10 and 25 μg mL−1) did not affect any measure of embryo development. All COCs treated with B. pelecinus fractions reached the final stage of embryo development, blastocyst hatching. Total blastocyst cell number was not affected. Conclusion The presence of fractions of B. pelecinus extract during in vitro oocyte maturation can reduce embryo development. Implications In vitro techniques can detect potential effects of forages on reproduction. Some fractions from an extract of B. pelecinus when present during oocyte maturation can reduce embryo development. The abundant PSC, loliolide, was not responsible. There was no indication that a PSC in B. pelecinus improves outcomes.

Histological Analysis of Somatic Embryogenesis from Immature Zygotic Embryo of Wild Banana Musa acuminata ssp. malaccensis

Somatic embryogenesis, a crucial plant regeneration method, has become indispensable for crop improvement, particularly for species reliant on somatic cell manipulation techniques. Optimization of this process necessitates an understanding of the developmental stages involved. This study investigates the histological aspects of somatic embryogenesis in Musa acuminata ssp. malaccensis derived from immature zygotic embryos. Through detailed histological analysis, we aimed to elucidate the morphological changes and cellular organization occurring during the various stages of somatic embryogenesis, from induction, culture proliferation, and somatic embryo development to plantlet conversion. The initial stages of embryogenesis, characterized by nodules, were primarily composed of meristematic cells with high cell division activity. These cells contained tetrad-like structures that could develop into distinct two- and four-celled proembryoids or proembryogenic aggregates. Our histo-anatomical analysis revealed that embryogenic cultures proliferated through multiple pathways simultaneously: somatic embryo budding, proembryo formation, and pro-embryonic mass formation from both internal and peripheral cells. At the stage of somatic embryo development, embryos with a well-defined protoderm layer, containing cells with prominent nuclei and dense cytoplasm, potentially regenerate into plantlets. Furthermore, histological examination revealed the presence of procambium within mature somatic embryos, which subsequently developed into the vascular system of the complete plantlet

Identify high-risk DOR women ≤ 35 years old following assisted reproduction technology through cutoffs of anti-mullerian hormone and antral follicle counts

BackgroundFemales with diminished ovarian reserve (DOR) have significantly lower cumulative live birth rates (CLBRs) than females with normal ovarian reserve. A subset of young infertile patients, whose ovarian reserve is declining but has not yet met the POSEIDON criteria for DOR, has not received the attention it merited. These individuals have not been identified in a timely manner prior to the initiation of assisted reproductive technology (ART), leading to suboptimal clinical pregnancy outcomes. We categorized this overlooked cohort as the “high-risk DOR” group.ObjectiveThe primary aim of this study was to identify high-risk DOR patients through anti-Mullerian hormone (AMH) and antral follicle counts (AFCs).MethodsA total of 10037 young women (≤ 35 years old) who underwent their first initial oocyte aspiration cycle at a single reproductive medicine center were included and further classified into three groups, based on the thresholds for AMH and AFC established through receiver operating characteristic (ROC) analysis and in alignment with the POSEIDON criteria. Two ROC analyses were performed to identify the cutoff values of AMH and AFC to obtain one viable embryo (one top-quality embryo or one viable blastocyst). The cutoffs of ROC were measured by sensitivity and specificity. The primary outcome was the cumulative live birth rate (CLBR) per oocyte aspiration cycle. The secondary outcomes included the number of oocytes retrieved and the number of viable embryos formed. Pearson’s chi-square tests were conducted to compare the clinical outcomes among the three groups. Furthermore, univariate logistic regression analyses were performed to investigate the associations between ovarian reserve and clinical outcomes. All of the above comparisons between the high-risk DOR and NOR were further confirmed by propensity score matching (PSM) (1:1 nearest-neighbor matching, with a caliper width of 0.02).ResultsAccording to the ROC analyses and POSEIDON criteria, the present study identified a population of high-risk DOR patients (1.20 ng/mL < AMH values < 2.50 ng/mL, with 6 ≤ AFC ≤ 10; n = 682), and their outcomes were further compared to those of DOR patients (positive control, AMH values ≤ 1.2 ng/mL, and/or AFC ≤ 5; n = 1153) and of NOR patients (negative control, 2.5 ng/mL ≤ AMH values ≤ 5.5 ng/mL, and 11 ≤ AFC ≤ 20; n = 2649). Patients in the high-risk DOR group had significantly lower CLBRs than those in the NOR group (p < 0.001) but higher CLBRs than those in the DOR group (p < 0.001). Logistic regression further demonstrated that high-risk DOR was associated with a lower likelihood of cumulative live birth chance (OR 0.401, 95% CI: 0.332–0.486, p < 0.001) than NOR was, with a greater likelihood of cumulative live birth chance (OR 1.911, 95% CI:1.558–2.344, p < 0.001) than DOR was. To investigate the effects of embryo development stage, the outcomes of D3 embryos and blastocysts were analyzed separately. Significant differences in pregnancy outcomes were detected only in D3 embryo ET cycles among the three groups (high-risk DOR vs. NOR, all p < 0.05; DOR vs. NOR, all p < 0.05). DOR/high-risk DOR did not influence the pregnancy loss rates or pregnancy outcomes (clinical pregnancy rates and ongoing pregnancy rates) per positive HCG cycle (all p > 0.05). After PSM, the differences in ovarian response and pregnancy outcomes between the high-risk DOR and NOR groups were consistent with the results before PSM.Conclusion(s)Our study revealed that the CLBR of the high-risk DOR patients was significantly lower than that of females with normal ovarian reserve and greater than that of females with DOR. The values of AMH ranging from 1.2 to 2.5 and AFC ranging from 6 to 10 appeared to constitute meaningful thresholds in females with mildly reduced ovarian reserve.

Pre-Embryonic Period Observation Shows a Unique Reproductive Strategy of the Critically Endangered Anji Salamander (Hynobius amjiensis).

Hynobius amjiensis, also known as the Anji salamander, is an amphibian species currently categorized as endangered due to its limited geographical distribution, primarily in China. To address the critical conservation status of this species, artificial breeding is essential for population expansion. However, progress in artificial breeding efforts has been hindered by the scarcity of research on the reproductive biology of the Anji salamander. In this study, we identified 25 distinct early stages of embryo development. Additionally, we observed that Anji salamander embryos contain a lesser amount of yolk compared to other salamanders or frogs. We further discovered that the Anji salamander employs a highly competitive reproductive strategy, producing a smaller number of high-quality offspring. This strategy aims to generate adaptive individuals through intense intraspecific competition, driven by three factors: extremely confined breeding habitats, a substantial number of eggs, and a reduced yolk content. We introduce the term "mass escape" to describe this strategy, which provides a novel perspective on cannibalization, focusing on the consumption of specific body parts rather than a single-individual activity. This study offers valuable insights into artificial breeding techniques designed to mitigate inherent intraspecific competitive pressure, thereby improving metamorphosis and survival rates. Additionally, it provides a comprehensive table detailing the pre-embryonic developmental stages of the Anji salamander.

Transcriptome analysis reveals the dynamics of gene expression during early embryonic development of hybrid groupers

Distant hybridization is widely applied in genetic improvement of aquatic animals. Although this technique should be suitable for genetic improvement of grouper, few studies related to offspring produced via distant hybridization have been reported. There we studied the gene expression dynamics of early embryonic development of groupers from self-breeding, intraspecific breeding and intergeneric hybridization. We first obtained offspring from female brown-marbled grouper (Epinephelus fuscoguttatus) and male leopard coral grouper (Plectropomus leopardus) and described the morphological characteristics of different developmental stages of embryos. We then constructed the transcriptome maps during early embryonic development of hybrid groupers. In addition, we also found that the process of MZT occurred from high blastula to early gastrula stage in the embryo of hybrid grouper and that wnt signaling pathway play an important role in dorsal-ventral axial formation during embryonic development of grouper. These results provide a foundation for the hybrid breeding of leopard coral grouper.

Elucidation of the pluripotent potential of bovine embryonic lineages facilitates the establishment of formative stem cell lines.

The establishment of epiblast-derived pluripotent stem cells (PSCs) from cattle, which are important domestic animals that provide humans with milk and meat while also serving as bioreactors for producing valuable proteins, poses a challenge due to the unclear molecular signaling required for embryonic epiblast development and maintenance of PSC self-renewal. Here, we selected six key stages of bovine embryo development (E5, E6, E7, E10, E12, and E14) to track changes in pluripotency and the dependence on signaling pathways via modified single-cell transcription sequencing technology. The remarkable similarity of the gene expression patterns between cattle and pigs during embryonic lineage development contributed to the successful establishment of bovine epiblast stem cells (bEpiSCs) using 3i/LAF (WNTi, GSK3βi, SRCi, LIF, Activin A, and FGF2) culture system. The generated bEpiSCs exhibited consistent expression patterns of formative epiblast pluripotency genes and maintained clonal morphology, normal karyotypes, and proliferative capacity for more than 112 passages. Moreover, these cells exhibited high-efficiency teratoma formation as well as the ability to differentiate into various cell lineages. The potential of bEpiSCs for myogenic differentiation, primordial germ cell like cells (PGCLCs) induction, and as donor cells for cellnuclear transfer was also assessed, indicating their promise in advancing cell-cultured meat production, gene editing, and animal breeding.

An efficient and easy-to-use protocol for induction of haploids in cucumber through parthenogenic embryo development

BackgroundCucumber (Cucumis sativus L.) is a model crop to study cell biology, including the development of haploids and doubled haploids in vegetable crops. In plant breeding, haploid and doubled haploids are valuable tools for developing pure homozygous inbred lines and accelerating genetic progress by reducing the time required for breeding cycles. Besides, the haploids are also valuable in genomic studies. We are reporting the induction of haploids in cucumber involving gynoecious and parthenocarpic genotypes for the first time. This study aimed to assess the efficient induction of haploids through pollination with gamma-irradiated pollen in cucumber. The effect of gamma irradiation dose on pollen viability and germination, fruit setting percentage, seed development, and haploid embryo development in cucumber hybrid genotypes were studied in detail. The goal was to utilize this information to produce haploid plants for genomics and transformation works in this model vegetable crop.ResultsPollination was done on six cucumber genotypes using varying doses of gamma rays (100, 200, 300, 400, and 500 Gy). Genotypes, doses of irradiation, and embryo developmental stage influenced the successful generation of in-vitro haploid plants. The optimal timeframe for embryo rescue was found to be 25 to 30 days after pollination. Haploid embryos were effectively induced using irradiated pollen at 400 to 500 Gy doses. Parthenogenetic plantlets were analyzed, and their ploidy level was confirmed through stomatal physiology, cytology (mitosis), and flow cytometry methods.ConclusionThrough parthenogenic embryo development, it is possible to induce a large number of haploids in cucumber. This technique’s power lies in its ability to streamline the breeding process, enhance genetic gain, and produce superior cultivars that contribute to sustainable agriculture and food security.

The effect of male factors on embryo morphokinetics: a retrospective analysis of 2726 blastocysts.

Male infertility may influence fertilization rates, embryo morphology, and implantation rates in in vitro fertilization (IVF) cycles. Oocyte competence plays a major role in embryo development, but there is a limited understanding of the connection between sperm quality, embryo development, and morphokinetic parameters using donor oocytes. The study evaluated if sperm quality may influence the morphokinetic parameters in IVF cycles. A retrospective multicentric observational cohort study included 747 ICSI cycles using donor oocytes with fresh or frozen sperm. Embryos were cultured in time-lapse incubators until the blastocyst stage. The population was divided into three groups according to sperm concentration, as control group (> 16 mill/mL), severe oligospermia (0-5 mill/mL), and moderate oligospermia group (5-16 mill/mL). Morphokinetic analysis showed no difference in the time from the 2-cell to 6-cell stage of embryo development. A significant difference was observed on day 3 of embryo development, specifically at the 7-cell stage (t7), severe oligospermia 53.37 ± 9.81, moderate oligospermia 56.95 ± 9.78, and control 55.1 ± 8.85h post-insemination (hpi) (p = 0.024), and 8-cell stage (t8), severe oligospermia 55.41 ± 10.83, moderate oligospermia 61.86 ± 12.38 hpi (p < 0.001), and control 58.61 ± 11.33. Accordingly, the synchrony of the four cleavages going from 4 to 8 cells (s3) was found statistically different among the groups in the severe oligospermia 8.05 ± 9.99, moderate oligospermia 11.66 ± 11.04 hpi, and control 8.55 ± 8.58 (p = 0.009). Morphokinetic time ranges were obtained for t6, t7, t8, and s3 in order to identify the good-quality blastocysts. Poor sperm quality is associated with alterations in morphokinetic parameters on day 3 in IVF cycles with donor oocytes, underlining the important role of spermatozoa during embryo development.

Effects of ovarian stimulation on embryo euploidy: an analysis of 12874 oocytes and 3106 blastocysts in cycles with preimplantation genetic testing for monogenic disorders.

Does ovarian stimulation and the ovarian response affect embryo euploidy? Ovarian stimulation and the ovarian response in women undergoing preimplantation genetic testing for monogenic disorders (PGT-M) cycles did not affect the rates of blastocyst euploidy. Whether or not ovarian stimulation in IVF-embryo transfer has potential effects on embryo euploidy is controversial among studies for several reasons: (i) heterogeneity of the study populations, (ii) biopsies being performed at different stages of embryo development and (iii) evolution of the platforms utilized for ploidy assessment. Patients who undergo PGT-M cycles typically have no additional risks of aneuploidy, providing an ideal study population for exploring this issue. A retrospective cohort study including embryos undergoing PGT-M was conducted at a single academically affiliated fertility clinic between June 2014 and July 2021. A total of 617 women with 867 PGT-M cycles involving 12874 retrieved oocytes and 3106 trophectoderm biopsies of blastocysts were included. The primary outcome of the study was median euploidy rate, which was calculated by dividing the number of euploid blastocysts by the total number of biopsied blastocysts for each cycle. Secondary outcomes included the median normal fertilization rate (two-pronuclear (2PN) embryos/metaphase II oocytes) and median blastulation rate (blastocyst numbers/2PN embryos). Comparable euploidy rates and fertilization rates were observed across all age groups, regardless of variations in ovarian stimulation protocols, gonadotropin dosages (both the starting and total dosages), stimulation durations, the inclusion of human menopausal gonadotrophin supplementation, or the number of oocytes retrieved (all P > 0.05). Blastulation rates declined with increasing starting doses of gonadotropins in women aged 31-34 years old (P = 0.005) but increased with increasing gonadotrophin starting doses in women aged 35-37 years old (P = 0.017). In women aged 31-34, 35-37, and 38-40 years old, blastulation rates were significantly reduced with increases in the number of oocytes retrieved (P = 0.001, <0.001, and 0.012, respectively). Limitations include the study's retrospective nature and the relatively small number of patients of advanced age, especially patients older than 40 years old, leading to quite low statistical power. Second, as we considered euploidy rates as outcome measures, we did not analyze the effects of ovarian stimulation on uniform aneuploidy and mosaicism, respectively. Finally, we did not consider the effects of paternal characteristics on embryo euploidy status due to the fact that blastocyst aneuploidy primarily originates from maternal meiosis. However, sperm factors might have an effect on embryo development and the blastulation rate, and therefore also the number of blastocysts analyzed. The exclusion of patients with severe teratozoospermia and the fact that only ICSI was used as the insemination technique for women undergoing PGT-M contributed to minimize the effect of paternal factors. Ovarian stimulation and response to stimulation did not affect blastocyst euploidy rates in women undergoing PGT-M cycles. However, in women aged 31-40 years old, there was a significant decline in blastulation rates as the number of retrieved oocytes increased. This study was supported by the National Natural Science Foundation of China (Grant No. 81701407, 82301826); the National Key Research and Development Program of China (2022YFC2702901, 2022YFC2703004); China Postdoctoral Science Foundation (2022M710261), and China Postdoctoral Innovation Talent Support Program (BX20220020). There is no conflict of interest. N/A.

Use of time-lapse technology on fertilization verification, embryo evaluation, and utilization: A national survey in Japan

BackgroundTime-lapse technology (TLT) has emerged as a significant advancement in the field of assisted reproductive technology (ART), providing continuous observation of embryos. However, limited information exists on the adoption of TLT across ART facilities and the clinical implications of its application in embryo evaluation and fertilization verification. The existing literature has not yet comprehensively examined how TLT data are utilized to optimize ART outcomes, particularly in Japan, where ART practices are highly prevalent. ObjectivesThis study aimed to investigate the adoption rate of TLT and its clinical effects on fertilization verification, embryo evaluation, and utilization of ART in Japan. Study DesignAn online survey was conducted from December 23, 2022, to January 16, 2023, in 616 ART facilities with both email and mailed notices. The survey investigated the utilization of TLT in each facility's evaluation of oocyte morphology, fertilization, embryo culture, and morphology. ResultsOverall, 345 responses were analyzed. Of these, only 42.6% confirmed fertilization at 16 to 18 hours after insemination. Most facilities defined normally fertilized eggs as 2 pronuclei (2PN; 53.3%) or a combination of a second polar body extrusion and 2PN (44.9%). Overall, 54.6% of the facilities had adopted TLT, and 76.9% to 96.9% of these facilities used TLT images for fertilization verification. At these centers, the use of 0PN embryos decreased, whereas the use of 2.1PN embryos increased. The rates of culture medium supplemented with antioxidants and hyaluronan were significantly higher in facilities with TLT than in those without TLT. TLT images were used for embryo evaluation in 94.3% of the facilities, while 31.0% used a combination of TLT images and artificial intelligence-based scoring systems. ConclusionsWhile TLT use is widespread in Japan, its application in evaluating fertilization and embryo development stages varies across facilities. Reaching a consensus on the optimal use of the TLT system will enhance the effectiveness, safety, and efficiency of ARTs.

MAT2A is essential for zygotic genome activation by maintaining of histone methylation in porcine embryos

Methionine adenosyltransferase 2A (MAT2A) is an essential enzyme in the methionine cycle that generates S-adenosylmethionine (SAM) by reacting with methionine and ATP. SAM acts as a methyl donors for histone and DNA methylation, which plays key roles in zygotic genome activation (ZGA). However, the effects of MAT2A on porcine ZGA remain unclear. To investigate the function of MAT2A and its underlying mechanism in porcine ZGA, MAT2A was knocked down by double-stranded RNA injection at the 1-cell stage. MAT2A is highly expressed at every stage of porcine embryo development. The percentages of four-cell-stage embryos and blastocysts were lower in the MAT2A-knockdown (KD) group than in the control group. Notably, depletion of MAT2A decreased the levels of H3K4me2, H3K9me2/3, and H3K27me3 at the four-cell stage, whereas MAT2A KD reduced the transcriptional activity of ZGA genes. MAT2A KD decreased embryonic ectoderm development (EED) and enhancer of zeste homolog 2 (EZH2) expression. Exogenous SAM supplementation rescued histone methylation levels and developmental arrest induced by MAT2A KD. Additionally, MAT2A KD significantly increased DNA damage and apoptosis. In conclusion, MAT2A is involved in regulating transcriptional activity and is essential for regulating histone methylation during porcine ZGA.

Antiviral RNA interference inhibits virus vertical transmission in plants

Known for over a century, seed transmission of plant viruses promotes trans-continental virus dissemination and provides the source of infection to trigger devastating disease epidemics in crops. However, it remains unknown whether there is a genetically defined immune pathway to suppress virus vertical transmission in plants. Here, we demonstrate potent immunosuppression of cucumber mosaic virus (CMV) seed transmission in its natural host Arabidopsis thaliana by antiviral RNA interference (RNAi) pathway. Immunofluorescence microscopy reveals predominant embryo infection at four stages of embryo development. We show that antiviral RNAi confers resistance to seed infection with different genetic requirements and drastically enhanced potency compared with the inhibition of systemic infection of whole plants. Moreover, we detect efficient seed transmission of a mutant CMV lacking its RNAi suppressor gene in mutant plants defective in antiviral RNAi, providing further support for the immunosuppression of seed transmission by antiviral RNAi.

Automatic ploidy prediction and quality assessment of human blastocysts using time-lapse imaging

Assessing fertilized human embryos is crucial for in vitro fertilization, a task being revolutionized by artificial intelligence. Existing models used for embryo quality assessment and ploidy detection could be significantly improved by effectively utilizing time-lapse imaging to identify critical developmental time points for maximizing prediction accuracy. Addressing this, we develop and compare various embryo ploidy status prediction models across distinct embryo development stages. We present BELA, a state-of-the-art ploidy prediction model that surpasses previous image- and video-based models without necessitating input from embryologists. BELA uses multitask learning to predict quality scores that are thereafter used to predict ploidy status. By achieving an area under the receiver operating characteristic curve of 0.76 for discriminating between euploidy and aneuploidy embryos on the Weill Cornell dataset, BELA matches the performance of models trained on embryologists’ manual scores. While not a replacement for preimplantation genetic testing for aneuploidy, BELA exemplifies how such models can streamline the embryo evaluation process.