Early Stages Of Embryo Development Research Articles

O-058 Newly identified female mutations causing oocyte maturation defect and embryo developmental arrest

Abstract Female infertility is a complex issue affecting a significant number of women. In the United States, among married women aged 15–49 years with no prior births, approximately one in five (19%) were unable to get pregnant after one year of trying. In addition, approximately one in four (26%) women in this group had difficulty getting pregnant or carrying a pregnancy to term. For women under 35 years of age, infertility is defined as a lack of success within one year, while for women aged 35 and older, it is within six months. Various factors, such as genetic, endocrine, physiological, anatomical, and immunological irregularities within the reproductive system, can affect a woman's chances of achieving pregnancy and delivering a healthy child. Among these factors, oocyte maturation is an important prerequisite for successful fertilization and subsequent embryonic development. In Medically Assisted Reproduction (MAR), the meiotic maturation of oocytes is induced by human chorionic gonadotropin (hCG) injection, which mimics the natural endogenous luteinizing hormone (LH) surge during the menstrual cycle. Although it is common for a few oocytes to remain immature despite ovarian stimulation and hCG administration, generally, immature oocytes have the competency to spontaneously mature in vitro. However, oocyte maturation is a sophisticated process involving multiple genes or protein molecules. Therefore, pathogenic variations in the genomic sequence or functional defects in any molecule that affects meiosis may lead to impaired oocyte maturation. While complete failure of all oocytes to mature in vivo is extremely rare, understanding the molecular aetiology of oocyte maturation is crucial for addressing infertility issues related to this process and improving fertility outcomes. The transition from the cleavage to the blastocyst stage often represents a significant bottleneck, with nearly 10% of fertilized eggs arrested at the cleavage stages. Developmental arrest of the preimplantation embryo is characterized by the cessation of cellular division for at least 24 h. Various factors originating from both embryonic and parental sources contribute to arrest of early stage embryo development. The genetic roots of developmental arrest were examined by considering both the parental causes of infertility and factors within the embryo. Examination revealed shared elements, regardless of the source of origin. Chromosomal abnormalities, abnormal preimplantation development, and monogenic variations have been reported to cause embryonic developmental arrests. Recently, deficiencies in oocyte maturation and embryo development have been categorized as part of the oocyte/zygote/embryo arrest (OZEMA) phenotype in the Online Mendelian Inheritance in Man (OMIM) database. A latest extensive review of monogenic causes associated with female infertility identified 23 genes with a total of 27 gene disease relationships (GDRs) for the OZEMA phenotype. Seventeen GDRs were classified as having at least moderate evidence of involvement in OZEMA, in addition to ten GDRs with less substantial evidence. Several additional genes were subsequently identified. This presentation will provide an overview of gene defects linked to the OZEMA phenotype, discuss the current status and future prospects of pre-MAR screening of females with OZEMA, and assess a novel workflow for modern genetic diagnosis to maximize the benefit of the patient.

The Blastulation Rate Is Negatively Associated With Euploid Rate

ObjectivesTo study the association between the blastulation rate, the presence of 1 pronucleus (1PN) zygotes, and the ploidy of the cohort of blastocysts. MethodsA cross-sectional study using the existing databases of 2 university fertility centres in Canada. We included 345 cycles from 235 couples who underwent next-generation sequencing preimplantation genetic testing for the detection of aneuploidy in the study. ResultsA total of 1456 blastocysts were biopsied. In multivariate analysis, only female age and the number of 1PN/2PN embryos showed a negative association with euploid ratio. Surprisingly, when the analysis was limited to cycles with no delayed blastulation, the blastulation rate was also negatively associated with the euploid ratio. ConclusionsThis study sheds some light on the stages of early embryo development. Further study on the mechanisms governing embryo development and the different cell cycle checkpoints in embryo development is warranted.

Inhibition of Atm and Atr kinases affects embryo development and the DNA damage response in sterlet (Acipenser ruthenus)

Embryo development is regulated by numerous interconnected pathways that direct cell fate, differentiation, and genetic stability. During development, these pathways can be altered by exogenous factors, such as pollutants, or endogenous factors, such as replication stress. Ataxia telangiectasia mutated (Atm) and ataxia telangiectasia and Rad3-related (Atr) kinases play critical roles in regulating the cell cycle, the DNA damage response (DDR), DNA repair, checkpoint activation, and apoptosis. In cells with damaged DNA, these kinases can slow the cell cycle to provide the necessary time and ability for DNA repair. In this study, we investigated the roles of Atm and Atr in embryo development and DDR in the sterlet (Acipenser ruthenus). Sterlets belong to the Acipenseridae family, one of the most threatened groups of species. Thus, understanding their genetics, biology, embryogenesis, and conservation is crucial. Furthermore, the sterlet is a significant aquaculture species that represents an intriguing model for studying polyploidy and genome plasticity. In our research, we examined the effects of chemical inhibition of Atm and Atr on sterlet embryo development in the absence and presence of the genotoxicant camptothecin (CPT). Our findings indicated that in the absence of genotoxic challenge, Atr autophosphorylation increases between 1 and 3 days post-fertilization (dpf) and decreases by 5 dpf, illustrating the involvement of Atr in the response to replication stress in rapidly differentiating tissues during the early stages of embryo development. Conversely, Atm inhibition was associated with a dose-dependent reduction in embryo viability, highlighting its importance in normal embryo development. When sterlet embryos were exposed to CPT, both Atm and Atr were involved in DDR and the activation of apoptosis. Notably, when both kinases were inhibited simultaneously, the embryos lost their ability to induce apoptosis and mitigate DNA damage, resulting in 100% embryonic mortality. These results suggest that Atm and Atr have distinct functions during normal embryo development, but they can partially complement each other in DDR.

Longitudinal profiling of human androgenotes through single-cell analysis unveils paternal gene expression dynamics in early embryo development.

How do transcriptomics vary in haploid human androgenote embryos at single cell level in the first four cell cycles of embryo development? Gene expression peaks at the fourth cell cycle, however some androcytes exhibit unique transcriptional behaviors. The developmental potential of an embryo is determined by the competence of the oocyte and the sperm. However, studies of the contribution of the paternal genome using pure haploid androgenotes are very scarce. This study was performed analyzing the single-cell transcriptomic sequencing of 38 androcytes obtained from 10 androgenote bioconstructs previously produced in vitro (de Castro et al., 2023). These results were analyzed through different bioinformatics software such as g: Profiler, GSEA, Cytoscape, and Reactome. Single cell sequencing was used to obtain the transcriptomic profiles of the different androcytes. The results obtained were compared between the different cycles studied using the DESeq2 program and functional enrichment pathways using g: Profiler, Cytoscape, and Reactome. A wave of paternally driven transcriptomic activation was found during the third-cell cycle, with 1128 upregulated and 225 downregulated genes and the fourth-cell cycle, with 1373 upregulated and 286 downregulated genes, compared to first-cell cycle androcytes. Differentially expressed routes related to cell differentiation, DNA-binding transcription, RNA biosynthesis and RNA polymerase II transcription regulatory complex, and cell death were found in the third and fourth with respect to the first-cell cycle. Conversely, in the fourth cell cycle, 153 downregulated and 332 upregulated genes were found compared with third cell cycle, associated with differentially expressed processes related to E-box binding and zinc finger protein 652 (ZNF652) transcription factor. Further, significant overexpression of LEUTX, PRAMEF1, DUXA, RFPL4A, TRIM43, and ZNF675 found in androgenotes, compared to biparental embryos, highlights the paternal contributions to zygote genome activation. All raw sequencing data are available through the Gene Expression Omnibus (GEO) under accessions number: GSE216501. Extrapolation of biological events from uniparental constructs to biparental embryos should be done with caution. Maternal and paternal genomes do not act independently of each other in a natural condition. The absence of one genome may affect gene transcription of the other. In this sense, the haploid condition of the bioconstructs could mask the transcriptomic patterns of the single cells. The results obtained demonstrated the level of involvement of the human paternal haploid genome in the early stages of embryo development as well as its evolution at the transcriptomic level, laying the groundwork for the use of these bioconstructs as reliable models to dispel doubts about the genetic role played by the paternal genome in the early cycles of embryo development. This study was funded by Instituto de Salud Carlos III (ISCIII) through the project 'PI22/00924', co-funded by European Regional Development Fund (ERDF); 'A way to make Europe'. F.D. was supported by the Spanish Ministry of Economy and Competitiveness through the Miguel Servet program (CPII018/00002). M.J.E. was supported by Instituto de Salud Carlos III (PI19/00577 [M.J.E.]) and FI20/00086. P.dC. was supported by a predoctoral grant for training in research into health (PFIS PI19/00577) from the Instituto de Salud Carlos III. All authors declare having no conflict of interest with regard to this trial.

Oxidative Effects in Early Stages of Embryo Development Due to Alcohol Consumption.

Alcohol, a widely consumed drug, exerts significant toxic effects on the human organism. This review focuses on its impact during fetal development, when it leads to a spectrum of disorders collectively termed Fetal Alcohol Spectrum Disorders (FASD). Children afflicted by FASD exhibit distinct clinical manifestations, including facial dysmorphism, delayed growth, and neurological and behavioral disorders. These behavioral issues encompass diminished intellectual capacity, memory impairment, and heightened impulsiveness. While the precise mechanisms underlying alcohol-induced fetal damage remain incompletely understood, research indicates a pivotal role for reactive oxygen species (ROS) that are released during alcohol metabolism, inciting inflammation at the cerebral level. Ethanol metabolism amplifies the generation of oxidant molecules, inducing through alterations in enzymatic and non-enzymatic systems responsible for cellular homeostasis. Alcohol consumption disrupts endogenous enzyme activity and fosters lipid peroxidation in consumers, potentially affecting the developing fetus. Addressing this concern, administration of metformin during the prenatal period, corresponding to the third trimester of human pregnancy, emerges as a potential therapeutic intervention for mitigating FASD. This proposed approach holds promise for ameliorating the adverse effects of alcohol exposure on fetal development and warrants further investigation.

Impact of Ionomycin on the Gene Expression of In vitro Fertilized Bovine Embryos

Background: The embryo genomic material used in this study came from an equal contribution of maternal and paternal genes. Gene expression during IVEP embryo development was controlled by several factors: the genetic makeup of the embryo, the epigenetic contributions and the culture materials. The aims of this research were to study the impact of ionomycin treatment on the sperm capacitation time and its impacts on the gene expression of some sensitive genes (Bcl-2, Bax, HSPB1, IGF1 and GDF9) during IVEP. Methods: The semen was placed in a sterile 15ml tube containing 5ml of capacitation medium and then split into three different experimental groups: the first group was the control; the second was the low-treated group, which contained sperm in a capacitation medium with 25 nM of ionomycin; the third group included 50 nM ionomycin. Result: The gene expression of BAX during early cleavage did not show any differences. However, in the late cleavage stage, the control group presented higher gene expression (P less than 0.05) than that in the two treated groups. The Bcl-2 genes of the control group showed higher gene expression (P less than 0.05) in both the early and late cleavage stages compared to the low-treated group. Gene expression of the HSP-1 gene was observed during early embryo development in the treated group (25 nM ionomycin). In conclusion, concentration 25 nM ionomycin showed higher gene expression in the early stages of embryo development also the best-quality embryos and gene expression results were found by studying gene expression. The transition from the maternal and paternal genome to the embryonic genome and gene expression during embryo development is controlled by several factors.

Dynamic methylation pattern of H19DMR and KvDMR1 in bovine oocytes and preimplantation embryos.

This study aimed to evaluate the epigenetic reprogramming of ICR1 (KvDMR1) and ICR2 (H19DMR) and expression of genes controlled by them as well as those involved in methylation, demethylation, and pluripotency. We collected germinal vesicle (GV) and metaphase II (MII) oocytes, and preimplantation embryos at five stages [zygote, 4-8 cells, 8-16 cells, morula, and expanded blastocysts (ExB)]. DNA methylation was assessed by BiSeq, and the gene expression was evaluated using qPCR. H19DMR showed an increased DNA methylation from GV to MII oocytes (68.04% and 98.05%, respectively), decreasing in zygotes (85.83%) until morula (61.65%), and ExB (63.63%). H19 and IGF2 showed increased expression in zygotes, which decreased in further stages. KvDMR1 was hypermethylated in both GV (71.82%) and MII (69.43%) and in zygotes (73.70%) up to morula (77.84%), with a loss of methylation at the ExB (36.64%). The zygote had higher expression of most genes, except for CDKN1C and PHLDA2, which were highly expressed in MII and GV oocytes, respectively. DNMTs showed increased expression in oocytes, followed by a reduction in the earliest stages of embryo development. TET1 was downregulated until 4-8-cell and upregulated in 8-16-cell embryos. TET2 and TET3 showed higher expression in oocytes, and a downregulation in MII oocytes and 4-8-cell embryo. We highlighted the heterogeneity in the DNA methylation of H19DMR and KvDMR1 and a dynamic expression pattern of genes controlled by them. The expression of DNMTs and TETs geneswas also dynamic owing to epigenetic reprogramming.

Embryological observations on seed abortion in Hibiscus syriacus L. and physiological studies on nutrients, enzyme activity and endogenous hormones

Under natural conditions, most Hibiscus syriacus L. individuals form very few mature seeds or the mature seeds that do form are of poor quality. As a result, seed yield is poor and seeds have low natural germinability. These phenomena strongly hinder utilization of the excellent germplasm resources of H. syriacus. The study has shown that pollen activity and stigma receptivity were high on the day of anthesis, and the pistils and stamens were fertile. Pollen release and stigma receptivity were synchronous. But in styles following self and cross-pollination, pollen tube abnormalities (distortion and twisting of the pollen tubes) and callose deposition were observed. Cross-pollinated pollen tubes elongated faster and fewer pollen tube abnormalities were observed compared with self-pollinated pollen tubes. And during embryo development, abnormalities during the heart-shaped embryo stage led to embryo abortion. Imbalance in antioxidant enzyme activities and low contents of auxin and cytokinin during early stages of embryo development may affect embryo development. Therefore, a low frequency of outcrossing and mid-development embryo abortion may be important developmental causes of H. syriacus seed abortion. Nutrient deficiencies, imbalance in antioxidant enzyme activities, and a high content of abscisic acid at advanced stages of seed development may be physiological causes of seed abortion.

Molecular characterizations and functional roles of NANOG in early development of porcine embryos

Nanog homeobox (NANOG) is the gateway to the pluripotent ground state in mouse embryonic stem cells and early embryos. However, understanding of the molecular signatures and functional characteristics of porcine NANOG remains limited. In this study, we analyzed the gene structure and sequence characteristics of porcine NANOG and found that the porcine NANOG gene is localized on chromosome 5, while NANOG sequence on chromosome 1 is the processed pseudogene. We explored the expression pattern of NANOG in porcine early embryos by immunofluorescence staining and Realtime-PCR and RNA-seq, the results showed that transcription of porcine NANOG commences at the 4-cell stage, while expression of the NANOG protein is initially observed in the inner cell mass of blastocysts. Furthermore, we identified a NANOG splicing variant in porcine early embryos, which maintain the overall structure of the original NANOG mRNA, except for a deletion of 38 base pairs in the second exon. To further investigate the function of NANOG in early embryo development in pigs, we employed siRNA-mediated deletion of the two specific transcripts on porcine zygotes. The results showed that blastocyst rate was significantly reduced after NANOG deleting. A significant decrease in the expression of DNA methylation-related gene DNMT3B was also observed in D3 embryo from the NANOG deleting group. In conclusion, the porcine NANOG gene, accompanied by a single-exon processed pseudogene, exhibits two transcripts and plays a pivotal role in the development of early-stage embryos.

Case Study in Infants with Cleft Lip and Palate Who Attended the Fundación Operación Sonrisa Consultation in The Period 2015-2019

Cleft lips and palate are considered congenital craniofacial malformations originating in the early stage of embryo development, due to an incomplete fusion of the medial nasal processes and maxillary processes. A multidisciplinary team is required to carry out the ideal treatment for these childhood patients. Aims: The objective of this study was meant to describe the casuistry of patients with cleft lip and palate in infants who attended the Operation Smile Venezuela Foundation in the period from 2015 to 2019. Subjects and Method: This was a non-experimental and descriptive field research. The sample included medical records from patients with cleft lip and palate that were attended since 2015 to 2019, having in account exclusion and inclusion criteria. A questionnaire was used as an instrument through the Google Form application with 34 closed questions of dichotomous answers. The variables studied were epidemiological characteristics such as: age, gender and origin, also we analyzed types of clefts lip and palate and whether they obtained surgical resolution or not, which were collected and evaluated according to our type of research and the objectives. Results: 618 medical records were analyzed, with an average of showing a prevalence in preschool age ( 40%) , followed by minor infants ( 29%) and the age with less prevalence was newborns (1%). In terms of gender there was an almost equal distribution, males (58%) and females (42%). Geographically there was a predominance in the Capital District (20%), Miranda (15%), Aragua (13%) and Monagas (12%) during the period studied. But states such as Amazonas, Barinas, Cojedes, Delta Amacuro, Lara and Nueva Esparta had the lowest percentage (0%). Of the cleft lip the most prevalent was the Complete left Unilateral, followed by the Incomplete Left Unilateral and the Complete Bilateral cleft lip. The Complete Bilateral and the Hard and Soft Posterior Palate Cleft were the most common. Conclusions: It is essential to determine the epidemiological variants, as well as to know the etiology of the cleft lip alveolus palatal and how to prevent them. Likewise, through this work, the activity that Operation Smile Venezuela Foundation carried out day by day is made known.

Fluid extraction from the left-right organizer uncovers mechanical properties needed for symmetry breaking.

Humans and other vertebrates define body axis left-right asymmetry in the early stages of embryo development. The mechanism behind left-right establishment is not fully understood. Symmetry breaking occurs in a dedicated organ called the left-right organizer (LRO) and involves motile cilia generating fluid-flow therein. However, it has been a matter of debate whether the process of symmetry breaking relies on a chemosensory or a mechanosensory mechanism (Shinohara et al., 2012). Novel tailored manipulations for LRO fluid extraction in living zebrafish embryos allowed us to pinpoint a physiological developmental period for breaking left-right symmetry during development. The shortest critical time-window was narrowed to one hour and characterized by a mild counterclockwise flow. The experimental challenge consisted in emptying the LRO of its fluid, abrogating simultaneously flow force and chemical determinants. Our findings revealed an unprecedented recovery capacity of the embryo to re-fil and re-circulate new LRO fluid. The embryos that later developed laterality problems were found to be those that had lower anterior angular velocity and thus less anterior-posterior heterogeneity. Next, aiming to test the presence of any secreted determinant, we replaced the extracted LRO fluid by a physiological buffer. Despite some transitory flow homogenization, laterality defects were absent unless viscosity was altered, demonstrating that symmetry breaking does not depend on the nature of the fluid content but is rather sensitive to fluid mechanics. Altogether, we conclude that the zebrafish LRO is more sensitive to fluid dynamics for symmetry breaking.

P-195 Can we predict blastulation, implantation and live birth in early stages of embryo development? Clinical validation of a classification algorithm based on automatic morphokinetic annotations

Abstract Study question Can a classification algorithm based on automatic annotations of early morphokinetic timings improve embryo selection in ICSI cycles? Summary answer The automatic scoring resulted significantly predictive of blastulation, implantation and live birth, especially when combined with morphological evaluation, but not of euploidy status. What is known already The Eeva Test® is an imaging system that automatically annotates morphokinetic parameters of the early embryo development. The latest version features the Xtend® algorithm, which classifies embryos on day 3 of development from highest (1) to lowest (5) potential to reach blastocyst stage, based on four parameters: the automatically recorded parameters P2 (t3-t2) and P3 (t4-t3), plus oocyte age and number of cells. Previous bibliography suggests that the Eeva scoring, in combination with morphologic evaluation, might also be predictive of implantation and live birth potential. However, the efficacy and reproducibility of this algorithm need to be validated with external data. Study design, size, duration Retrospective, observational cohort study, performed over 3736 embryos from oocyte donation cycles and 1291 embryos from autologous cycles with preimplantation genetic testing for aneuploidies (PGT-A) performed in a single IVF clinic over 3 years. All embryos were scored by the Eeva Test on day 3. The performance of the Eeva-Xtend scoring system as predictor of blastocyst development, euploidy, implantation and live birth was assessed. Participants/materials, setting, methods Embryo development was monitored by a time-lapse system. Embryo selection was performed by morphological (ASEBIR) criteria, resulting in the transfer of 959 embryos. The correlation between the Eeva-Xtend scoring, individually and combined with morphological classification, and the study outcomes was quantified by generalized estimating equations (GEE) including main possible confounders, and expressed in terms of odds ratio (OR). The performance of the GEEs was quantified and compared through the Area Under the ROC Curves (AUCs). Main results and the role of chance A positive association (P<0.001) was confirmed between lower Eeva scores and higher odds of reaching blastocyst stage (OR (1 vs 5) = 15.849, 95%CI 12.510-20.078; OR (2 vs 5) = 11.592, 95%CI 9.229-14.560; AUC = 0.768 (95%CI 0.754−0.783)), and consistent in both oocyte donation and PGT-A cycles. Lower morphokinetic embryo scores also resulted significantly associated with higher chances of implantation (OR (1 vs 5) = 3.385, 95%CI 1.507-7.605; P = 0.003) and live birth (OR (1 vs 5) = 5.132, 95%CI 2.089-12.605; P<0.001) in oocyte donation cycles, but not in embryos subjected to PGT-A: OR (1 vs 5) = 2.089, 95%CI 0.321-13.614, P = 0.441 for implantation and OR (1 vs 5) = 0.916, 95%CI 0.161-5.205, P = 0.921 for live birth. In embryos subjected to PGT-A, the automatic scoring system did not result significantly associated with the euploidy status: OR (1 vs 5) = 0.755 (95%CI 0.255−0.981; P = 0.489). In global, the Eeva Test had similar performance than morphological evaluation for implantation (AUC = 0.610 (95%CI 0.572−0.648) vs 0.606 (95%CI 0.568−0.644), respectively) and live birth (AUC = 0.622 (95%CI0.584−0.660) vs 0.609 (95%CI 0.571−0.647)) prediction. However, the highest performance was achieved when combining both scoring systems: AUC for implantation = 0.629 (95%CI 0.592−0.666), AUC for live birth = 0.636 (95%CI 0.598−0.673). Limitations, reasons for caution The retrospective nature of the study introduces some variability in the characteristics of the sample. However, the large sample size partially overcomes said limitation. The automatic annotations of the morphokinetic parameters were not validated in this study. Wider implications of the findings Our results support the applicability and reproducibility of this automatic early embryo evaluation system. Furthermore, our findings support that the use of this automatic embryo evaluation system in combination with morphological evaluation can potentially reduce subjectivity, improve embryo evaluation and increase success rates in IVF treatments. Trial registration number not applicable

Extracellular Vesicles Secreted by Pre-Hatching Bovine Embryos Produced In Vitro and In Vivo Alter the Expression of IFNtau-Stimulated Genes in Bovine Endometrial Cells.

The embryo-maternal interaction occurs during the early stages of embryo development and is essential for the implantation and full-term development of the embryo. In bovines, the secretion of interferon Tau (IFNT) during elongation is the main signal for pregnancy recognition, but its expression starts around the blastocyst stage. Embryos release extracellular vesicles (EVs) as an alternative mechanism of embryo-maternal communication. The aim of the study was to determine whether EVs secreted by bovine embryos during blastulation (D5-D7) could induce transcriptomic modifications, activating IFNT signaling in endometrial cells. Additionally, it aims to assess whether the EVs secreted by embryos produced in vivo (EVs-IVV) or in vitro (EVs-IVP) have different effects on the transcriptomic profiles of the endometrial cells. In vitro- and in vivo-produced bovine morulae were selected and individually cultured for 48 h to collect embryonic EVs (E-EVs) secreted during blastulation. E-EVs stained with PKH67 were added to in vitro-cultured bovine endometrial cells to assess EV internalization. The effect of EVs on the transcriptomic profile of endometrial cells was determined by RNA sequencing. EVs from both types of embryos induced several classical and non-classical IFNT-stimulated genes (ISGs) and other pathways related to endometrial function in epithelial endometrial cells. Higher numbers of differentially expressed genes (3552) were induced by EVs released by IVP embryos compared to EVs from IVV (1838). Gene ontology analysis showed that EVs-IVP/IVV induced the upregulation of the extracellular exosome pathway, the cellular response to stimulus, and the protein modification processes. This work provides evidence regarding the effect of embryo origin (in vivo or in vitro) on the early embryo-maternal interaction mediated by extracellular vesicles.

Enhanced proliferation in a prawn embryonic primary cell culture ectopically expressing mutated Ras

Crustacean cell line immortalization has gained a great deal of attention in recent decades for both scientific and applied reasons. Our goal in this study was to advance the state of art towards establishing an immortalized cell line by improving the proliferation rates of primary cells isolated from embryos of the giant freshwater prawn Macrobrachium rosenbergii by using a lentivirus expressing the Ras oncogene. The choice of Ras derived from its involvement in various cellular pathways, such as cell growth, differentiation, and survival, and its use as a tool for in-vitro immortalization, e.g., a specific mutated Ras (RasV12) was used to generate an arthropod cell line. Complementarily, in-silico screening of M. rosenbergii transcriptomic libraries for Ras expression indicated that Ras is already expressed at very early stages of embryo development. In the current study, we transduced primary M. rosenbergii embryonic cells with a lentivirus expressing RasV12 by using the white spot syndrome virus (WSSV IE1) promoter. Expression and sequencing (as followed by sequencing cDNA, confocal microscopy and FACS analysis) of the mutated Ras in the transduced cells confirmed that the lentivirus was successfully integrated into the genome. The lenti-MrRas transduction rate was 23% in the total primary cell population and more than 80% in a sub-population of cells with high granularity. Proliferation of lenti-MrRas transfected cells was enhanced to almost 1200% of the seeding density by the end of our experiment (18 days), which was double that of the control. We were thus successful in enhancing the longevity of embryonic primary cell cultures by ectopic expression of the mutated Ras protein, but the improvement was not sufficient for immortalization.

Transcriptome changes of Apis mellifera female embryos with fem gene knockout by CRISPR/Cas9

The sex of honey bees is decided by a regulatory cascade comprising of csd, fem and Amdsx. In order to further identify other genes involved in sex determination and differentiation of honey bees in the early stages of embryo development, the CRISPR/Cas9 method was used to knock out fem gene in the embryonic stage of diploid western honey bees, and RNA-seq was used to analyze gene expression changes in the embryo after fem knockout. Finally, we found that the bees had undergone gender changes due to fem knockout. A total of 155 differentially expressed genes (DEGs) were obtained, with 48 up-regulated and 107 down-regulated DEGs in the mutant group compared to the control group. Of them, many genes are related to sex development or differentiation. In addition, 1502 differentially expressed alternative splicing events (DEASEs) related to 1011 genes, including the main honey bee sex-determining genes csd, tra2, fem, and Amdsx, were identified between the mutant group and control group, indicating that fem regulates alternative splicing of a large number of downstream genes. Our results provide valuable clues for further investigating the molecular mechanism of sex determination and differentiation in honey bees.

Oocyte maturity, oocyte fertilization and cleavage-stage embryo morphology are better in natural compared with high-dose gonadotrophin stimulated IVF cycles

Research questionDoes high-dose gonadotrophin stimulation have an effect on oocyte and early-stage embryo development? DesignThis was a retrospective study including 616 natural cycle IVF (NC-IVF) and 167 conventional IVF (cIVF) cycles. In total, 2110 oocytes were retrieved and analysed in fresh cycles. In NC-IVF, only human chorionic gonadotrophin was applied to trigger ovulation. In cIVF, antagonist protocols with daily 150–300 IU of human menopausal gonadotrophins were performed. The effect of gonadotrophins on oocyte and early-stage embryo development was analysed. Primary outcomes were the occurrence of mature (metaphase II) oocytes, zygotes and embryos with good morphology at the cleavage stage 2 days after oocyte retrieval. ResultsThe mature oocyte rate (number of mature oocytes/number of retrieved oocytes) was higher in NC-IVF than cIVF cycles (89% versus 82%, adjusted odds ratio [aOR] 1.79, P = 0.001), as was the zygote rate per oocyte retrieved (70% versus 58%, aOR 1.76, P = 0.001) and the zygote rate per mature oocyte (79% versus 71%, aOR 1.62, P = 0.001). The percentage of zygotes that developed into cleavage-stage embryos was no different. For the transferred embryos, the probability of having a good embryo morphology with four blastomeres and a fragmentation of <10% (score 0) in cleavage-stage embryos was found to be higher in NC-IVF (proportional aOR for four blastomeres 2.00, P < 0.001; aOR 1.87 for a fragmentation score of 0, P = 0.003). ConclusionsOocyte maturity, oocyte fertilization and morphology of the cleavage-stage embryo are affected by high-dose gonadotrophin stimulation in fresh IVF cycles.

Network analysis reveals different hub genes and molecular pathways for pig in vitro fertilized early embryos and parthenogenotes.

Maternal-to-zygotic transition (MZT) occurs when maternal transcripts decay and zygotic genome is activated gradually at the early stage of embryo development. Previously, single-cell RNA-seq (scRNA-seq) has helped us to uncover the MZT-associated mRNA dynamics of in vitro-produced pig early embryos. Here, to further investigate functional modules and hub genes associated with MZT process, the weighted gene co-expression network analysis (WGCNA) was performed on our previously generated 45 scRNA-seq datasets. For the in vitro fertilized embryo (IVF) group, 5 significant modules were identified (midnight blue/black/red and blue/brown modules, positively correlated with 1-cell (IVF1) and 8-cell (IVF8), respectively), containing genes mainly enriched in signalling pathways such as Wnt, regulation of RNA transcription, fatty acid metabolic process, poly(A) RNA binding and lysosome. For the parthenogenetically activated embryo (PA) group, 9 significant modules were identified (black/purple/red, brown/turquoise/yellow, and magenta/blue/green modules, positively correlated with MII oocytes, 1-cell (PA1) and 8-cell (PA8), respectively), mainly enriched in extracellular exosome, poly(A) RNA binding, mitochondrion and transcription factor activity. Moreover, some of identified hub genes within 3 IVF and 9 PA significant modules, including ADCY2, DHX34, KDM4A, GDF10, ABCC10, PAFAH2, HEXIM2, COQ9, DCAF11, SGK1 and ESRRB, have been reported to play vital roles in different biological processes. Our findings provide information and resources for subsequent in-depth study on the regulation and function of MZT in pig embryos.

P-124 The impact of Fragile x premutation carrier state on embryo morphokinetic development, A comparison between genetically normal and abnormal embryos

Abstract Study question Does inheritance of FMR1 pre mutant allele affect embryos morphokinetic development? Summary answer Embryos that inherit the FMR1 pre-mutant allele are of a lower morphokinetic quality at the blastocyte stage compared to those with the normal allele. What is known already Previous studies suggest lower oocyte yield and blastulation rate in FMR1 premutation carriers undergoing preimplantation genetic testing for monogenic diseases (PGT-M). Yet, data is lacking concerning embryo morphokinetic development in this group. Study design, size, duration Retrospective analysis, on 529 embryos from 126 in vitro fertilization (IVF)- PGT-M cycles of 39 FMR-1 premutation women carriers. Participants/materials, setting, methods Morphological and morphokinetic parameters acquired by the time-lapse monitoring system were compared between embryos carrying the FMR-1 permutated allele (FMR1 group n = 271) to those who inherited the normal allele (Normal group n = 258). Outcomes measures were embryos morphokinetic parameters up to day 3, the start of blasulation time (tSB) for day 5 embryos, and the rate of top-quality embryos at days 3 and 5. Main results and the role of chance No differences were found between the groups in all morphokinetic parameters from the time of ICSI until biopsy on day 3. Blastulation rate was comparable between the groups. However, FMR1 embryos exhibited delayed start of blastulation compared to the genetically normal embryos (median tSB 104.2 hrs (99.3-110.3) vs 101.6 hrs (94.5-106.7), P = 0.01) and had lower top quality embryo rate (25.6% vs 38.8%, P = 0.04). Limitations, reasons for caution This study is limited by its retrospective design and inability to assess CGG expansion in the embryo. Wider implications of the findings This study offers new insight into the impact of permutated FMR1 gene in the early stages of embryo development. Further studies are needed in order to apply these results in clinical decision-making. Trial registration number not applicable

P-252 Oolemma surface area impacts human pre-implantation embryo development and usage: The Rotterdam Periconception Cohort

Abstract Study question Does oolemma area change before the 2-cell stage? Is oolemma area associated with fertilization rate, embryo usage rate and preimplantation embryo development? Summary answer Oolemma area is associated with preimplantation embryo development, the odds of fertilizing and developing into a used or discarded embryo. What is known already Current morphological grading systems are based on embryo morphology and omit oocyte morphology. Fertilization and embryo potential are largely determined by the maturation and quality of the oocyte. Oocyte quality correlates to morphology, and the very early stages of embryo development are critical to changes in oocyte size. Larger oocyte size associates with higher quality blastocyst. This suggests that oocyte size can be a morphological marker of preimplantation embryo quality and assisted reproductive treatment (ART) outcomes. Study design, size, duration This study is embedded in the Rotterdam Periconceptional Cohort, an ongoing prospective tertiary hospital-based birth cohort study. From May 2017 to July 2020, a subcohort of 378 women that underwent ART was included comprising of 124 pregnancies after in vitro fertilization (IVF) and 254 after intracytoplasmic sperm injection (ICSI). Participants/materials, setting, methods Health, lifestyle and treatment factors were collected from detailed self-reported questionnaires and medical records. Oolemma area was measured at the time of fertilization (t0), pronuclear appearance (tPNa) and fading (tPNf). Oolemma area trajectories (slope surface t0-tPNf) were calculated with linear regression. Fertilization rate, embryo usage rate, and embryo morphokinetics were analyzed in association with oolemma area and trajectories. Analyses were performed with linear mixed models, mixed effects logistic regression, and adjusted for relevant confounders. Main results and the role of chance Oolemma area decreased from t0 to tPNf for oocytes that developed into both used or discarded embryos. Oolemma area at t0 was strongly associated with the rate of area shrinking, as larger oolemma areas had faster shrinking rates (Bshrinking -12.55 µm²/hour, 95%CI -14.55; -10.55, p &amp;lt; 0.001). Oocytes that resulted in a usable embryo were larger and reached tPNf faster than oocytes that resulted in a discarded embryo (tPNf 9855±595 µm2 vs 9690±721 µm2, p &amp;lt; 0.001, 24.4±3.6 h vs 26.5±5.8 h, p &amp;lt; 0.001). Oolemma area at tPNf did not differ between ICSI and IVF oocytes, however ICSI oocytes reached tPNf faster than IVF oocytes (23.6±3.2h and 25.9±3.8h, p &amp;lt; 0.001, respectively). In ICSI only, larger oolemma area showed lower odds of a successful fertilization (t0 OR 0.57, 95%CI 0.48; 0.67, p &amp;lt; 0.001), but higher odds of being cryopreserved or transferred (tPNf OR 1.66, 95%CI 1.33; 2.08, p &amp;lt; 0.001). For IVF and ICSI, larger oolemma area at tPNF was associated with faster preimplantation development up to the morula stage (t7 B -1.41, 95%CI -2.18; -0.63, p &amp;lt; 0.001). Oolemma area shrinking rate was not associated with embryo morphokinetics. Limitations, reasons for caution Small number of embryos measured at the blastocyst stage, and oolemma area measurements were not possible at t0 and tPNa for IVF oocytes. Wider implications of the findings Oolemma area is a suggested morphological marker for oocyte competence and an early marker for the success of fertilization and embryo development. The relevance of oolemma area as predictor of ART success requires further research. Cryopreservation and PCOS, which influence oocyte size, could be studied in association with oolemma area. Trial registration number not applicable

Combined effects of S-metolachlor and benoxacor on embryo development in zebrafish (Danio rerio)

It is necessary to study the combined toxicity of an herbicide and its safener because the two are often used in combination. S-metolachlor and its safener benoxacor have been detected in aquatic environments and can individually damage the oxidative stress system in zebrafish embryos (Danio rerio). However, only their separate toxicity in zebrafish (Danio rerio) embryo development has been reported. This study assessed the combined toxicity of benoxacor and S-metolachlor in zebrafish embryo development, including acute toxicity, developmental toxicity, oxidative damage, and cell apoptosis. The 96-h LC50 values were higher in mixtures of benoxacor and S-metolachlor than in benoxacor alone. The treatments included S-metolachlor, Mix-1 (0.1 mg/L benoxacor + 0.1 mg/L S-metolachlor), Mix-2 (0.1 mg/L benoxacor + 0.3 mg/L S-metolachlor) and benoxacor alone. Embryos exposed to Mix-1 and Mix-2 had lower developmental toxicities, superoxide dismutase (SOD) activity, osx and cat expression levels than those exposed to benoxacor alone. Moreover, glutathione S-transferase (GST), catalase (CAT), glutathione reductase (GR), glutathione peroxidase (GPx) activities, and the expressions of tbx16, nrf2, bcl2, and caspase9 were higher in the mixtures than in the benoxacor group. High-throughput RNA sequencing revealed that benoxacor had a greater effect on gene regulation than Mix-1 and Mix-2. The malformation rate, different enrichment gene numbers, and gene expression levels of hatched embryos were higher in Mix-1 than in Mix-2. The results indicate that a mixture of S-metolachlor and benoxacor has antagonistic effects in the early stage of embryo development. The mixtures can break the reactive oxygen species balance, causing abnormal cell apoptosis and developmental malformation in embryos. Besides investigating the combined toxicity of benoxacor and S-metolachlor in zebrafish embryo development, this study provides a risk assessment basis for a herbicide combined with its safener.