Early Human Embryonic Development Research Articles

Distinct dynamics of parental 5-hydroxymethylcytosine during human preimplantation development regulate early lineage gene expression.

The conversion of DNA 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) by TET enzymes represents a significant epigenetic modification, yet its role in early human embryos remains largely unknown. Here we showed that the early human embryo inherited a significant amount of 5hmCs from an oocyte, which unexpectedly underwent de novo hydroxymethylation during its growth. Furthermore, the generation of 5hmC in the paternal genome after fertilization roughly followed the maternal pattern, which was linked to DNA methylation dynamics and regions of sustained methylation. The 5hmCs persisted until the eight-cell stage and exhibited high enrichment at OTX2 binding sites, whereas knockdown of OTX2 in human embryos compromised the expression of early lineage genes. Specifically, the depletion of 5hmC affected the activation of embryonic genes, which was further evaluated by ectopically expressing mouse Tet3 in human early embryos. These findings revealed distinct dynamics of 5hmC and unravelled its multifaceted functions in early human embryonic development.

In vitro culture of cynomolgus monkey embryos from blastocyst to early organogenesis.

Human early embryonic development is the cornerstone of a healthy baby. Abnormal early embryonic development may lead to developmental and pregnancy-related disorders. Accordingly, understanding the developmental events and mechanisms of human early embryonic development is very important. However, attempts to reveal these events and mechanisms are greatly hindered by the extreme inaccessibility of in vivo early human embryos. Fortunately, the emergence of in vitro culture (IVC) systems for mammalian embryos provides an alternative strategy. In recent years, different two-dimensional and three-dimensional IVC systems have been developed for human embryos. Ethical limitations restrict the IVC of human embryos beyond 14 days, which makes non-human primate embryos an ideal model for studying primate developmental events. Different culture systems have supported the development of monkey embryos to days postfertilization 14 and 25, respectively. The successful recapitulation of in vivo developmental events by these IVC embryos has greatly enriched our understanding of human early embryonic development, which undoubtedly helps us to develop possible strategies to predict or treat various gestation-related diseases and birth defects. In this protocol, we establish different two-dimensional and three-dimensional IVC systems for primate embryos, provide step-by-step culture procedures and notes, and summarize the advantages and limitations of different culture systems. Replicating this protocol requires a moderate level of experience in mammalian embryo IVC, and the embryo culture requires strict adherence to the procedures we have described.

Combining single-cell profiling and functional analysis explores the role of pseudogenes in human early embryonic development

More and more studies have demonstrated that pseudogenes possess coding ability, and the functions of their transcripts in the development of diseases have been partially revealed. However, the role of pseudogenes in maintenance of normal physiological states and life activities has long been neglected. Here we identify pseudogenes that are dynamically expressed during human early embryogenesis, showing different expression pattern from that of adult tissues. We explore the expression correlation between pseudogenes and the parent genes, part due to their shared gene regulatory elements or the potential regulation network between them. The essential role of three pseudogenes, PI4KAP1, TMED10P1, and FBXW4P1, in maintaining self-renewal of human embryonic stem cells is demonstrated. We further find that the three pseudogenes might perform their regulatory functions by binding to proteins or microRNAs. The pseudogene-related single-nucleotide polymorphisms are significantly associated with human congenital disease, further illustrating their importance during early embryonic development. Overall, this study is an excavation and exploration of functional pseudogenes during early human embryonic development, suggesting that pseudogenes are not only capable of being specifically activated in pathological states, but also play crucial functions in the maintenance of normal physiological states.

O-072 The canonical Hippo signalling pathway controls the first lineage segregation in the human preimplantation embryo

Abstract Study question What is the role of the canonical Hippo signalling pathway during the first lineage segregation in the human preimplantation embryo? Summary answer Canonical Hippo signalling regulates the first lineage segregation by controlling the expression of trophectoderm (TE) and inner cell mass (ICM) associated genes. What is known already The Hippo signalling pathway is a conserved regulatory network that plays a critical role in the first lineage segregation in mouse, rat, bovine and human preimplantation embryos. This pathway consists of two distinct branches (canonical, non-canonical). Despite their shared role in lineage segregation, the respective functions of canonical and non-canonical Hippo signalling branches remain largely unexplored. Since these branches are regulated by distinct upstream mechanisms, they may have different roles in embryonic growth, function, and survival. Unravelling the distinct roles of these branches is essential for understanding the intricate mechanisms that govern early human embryonic development. Study design, size, duration This experimental study involved the functional characterization of the canonical Hippo signalling pathway by pharmacologically inhibiting the core kinase MST1/2. Eight-cell stage human embryos donated for research were warmed and incubated with or without the inhibitor, till completion of the first lineage segregation days post fertilization (dpf) 5. The control (n = 20) and treated embryos (n = 17) were evaluated based on embryonic development (kinetics, morphology), and expression of lineage-specific markers. Participants/materials, setting, methods The optimal concentration (1μΜ) of the highly selective XMU-MP-1 inhibitor targeting the MST1/2 core kinase of the canonical Hippo signalling pathway was established in titration experiments. Time-lapse imaging (Miri TL, Esco Medical) captured effects on embryonic development (kinetics, morphology). Changes in the main effectors of the Hippo signalling pathway (YAP1, p-YAP1 and WWTR1) and the expression of lineage-specific markers (ICM: NANOG, SOX2; TE: GATA3) were assessed through immunofluorescence imaging and confocal microscopy (LSM800, Zeiss). Main results and the role of chance For the first time, we provide a complete spatiotemporal analysis of the main effectors of the Hippo signalling pathway YAP1 and WWTR1. They are co-localised in the nuclei of outer cells in compacting (dpf3) and compacted (dpf4) embryos. In the dpf5 blastocyst, YAP1 and WWTR1 are detected in the nuclei of the TE cells. Phosphorylated effector p-YAP1, indicative of active Hippo signalling, was first detected in the inner cells of the compacted embryo (dpf4) and remained in the ICM of the dpf5 blastocyst, supporting the role of the Hippo signalling pathway in the first lineage segregation. Treatment of human embryos with XMU-MP-1 reduced p-YAP1 levels, confirming its specificity for MST1/2. Inhibition of the MST1/2 kinase had a significant effect on the developmental kinetics as treated embryos had lower blastocyst formation rates. Embryo morphology was significantly compromised as treated embryos formed ICMs with less cells compared to control embryos. Immunofluorescence analysis revealed that the XMU-MP-1 treatment led to the upregulation of YAP1 and upregulation of TE marker GATA3, while the ICM markers SOX2 and NANOG were downregulated. Overall, our data suggest that active Hippo signaling promotes ICM formation, while TE formation requires inactive Hippo signaling. Limitations, reasons for caution Caution is warranted due to potential non-specific effects of the XMU-MP-1 inhibitor, although this compound has been shown in other models to be very specific. Further studies should explore off-target impacts to ensure result reliability. Wider implications of the findings Understanding the signalling pathways that regulate human preimplantation embryo development is fundamental for understanding embryonic growth, function, and survival, and can also help improve IVF success rates by addressing deficiencies that lead to embryo developmental arrest. Trial registration number Not applicable

Influence of Long Non-Coding RNAs on Human Oocyte Development.

Recent research findings have highlighted the pivotal roles played by lncRNAs in both normal human development and disease pathogenesis. LncRNAs are expressed in oocytes and early embryos, and their expression levels change dynamically once the embryonic genome is activated during early human embryonic development. Abnormal expression of lncRNAs was found in follicular fluid, granulosa cells and oocytes of patients, and these lncRNAs were related to cell proliferation and apoptosis, nuclear maturation and follicle development. The expression levels of some lncRNAs in cumulus cells demonstrate correlations with the quality of oocytes and early embryos. This paper aims to present a comprehensive overview of the influence of LncRNAs on the developmental process of human oocytes as well as their involvement in certain infertility-related diseases.

Assessment and Comparison of Early Developmental Toxicity of Six Per- and Polyfluoroalkyl Substances with Human Embryonic Stem Cell Models.

Per- and polyfluoroalkyl substances (PFAS) are extensively utilized in varieties of products and tend to accumulate in the human body including umbilical cord blood and embryos/fetuses. In this study, we conducted an assessment and comparison of the potential early developmental toxicity of perfluorooctanoic acid (PFOA), undecafluorohexanoic acid (PFHxA), heptafluorobutyric acid, perfluorooctanesulfonate (PFOS), perfluorohexanesulfonate, and perfluorobutyric acid at noncytotoxic concentrations relevant to human exposure using models based on human embryonic stem cells in both three-dimensional embryoid body (EB) and monolayer differentiation configurations. All six compounds influenced the determination of cell fate by disrupting the expression of associated markers in both models and, in some instances, even led to alterations in the formation of cystic EBs. The expression of cilia-related gene IFT122 was significantly inhibited. Additionally, PFOS and PFOA inhibited ciliogenesis, while PFOA specifically reduced the cilia length. Transcriptome analysis revealed that PFOS altered 1054 genes and disrupted crucial signaling pathways such as WNT and TGF-β, which play integral roles in cilia transduction and are critical for early embryonic development. These results provide precise and comprehensive insights into the potential adverse health effects of these six PFAS compounds directly concerning early human embryonic development.

Morphokinetic features in human embryos: Analysis by our original high-resolution time-lapse cinematography-Summary of the past two decades.

The pioneering work by Dr. Payne etal. in time-lapse cinematography for observation of the morphokinetic features of human embryos inspired us to develop a new invitro culture system with high-resolution time-lapse cinematography (hR-TLC) back in 2001. This invitro culture system was capable of maintaining stable culture and was constructed on an inverted microscope stage. Embryos were observed and photographed noninvasively for an extended period, up to 7 days. The obtained images were displayed at a speed of 30 frames per second and individually analyzed. Using hR-TLC, human fertilization and subsequent embryonic development were visualized, revealing the time course of phenomena and many unusual dynamics. In this review, we summarize the results of our hR-TLC analysis of early human embryonic development over the past 20 years. In the near future, it is expected that the vast amount of information obtained by hR-TLC will be integrated into the AI system for further analysis and to provide feedback that will have the potential to improve clinical practice. In the era of SDGs and environmental awareness, we should be cautious about the direction in which AI can be utilized to avoid any further harm to the planet.

A DNA methylation haplotype block landscape in human tissues and preimplantation embryos reveals regulatory elements defined by comethylation patterns.

DNA methylation and associated regulatory elements play a crucial role in gene expression regulation. Previous studies have focused primarily on the distribution of mean methylation levels. Advances in whole-genome bisulfite sequencing (WGBS) have enabled the characterization of DNA methylation haplotypes (MHAPs), representing CpG sites from the same read fragment on a single chromosome, and the subsequent identification of methylation haplotype blocks (MHBs), in which adjacent CpGs on the same fragment are comethylated. Using our expert-curated WGBS data sets, we report comprehensive landscapes of MHBs in 17 representative normal somatic human tissues and during early human embryonic development. Integrative analysis reveals MHBs as a distinctive type of regulatory element characterized by comethylation patterns rather than mean methylation levels. We show the enrichment of MHBs in open chromatin regions, tissue-specific histone marks, and enhancers, including super-enhancers. Moreover, we find that MHBs tend to localize near tissue-specific genes and show an association with differential gene expression that is independent of mean methylation. Similar findings are observed in the context of human embryonic development, highlighting the dynamic nature of MHBs during early development. Collectively, our comprehensive MHB landscapes provide valuable insights into the tissue specificity and developmental dynamics of DNA methylation.

High-throughput sequencing reveals hub genes for human early embryonic development arrest in vitro fertilization: a pilot study.

Many clinical studies have shown that embryos of in vitro fertilization (IVF) are often prone to developmental arrest, which leads to recurrent failure of IVF treatment. Early embryonic arrest has always been an urgent clinical problem in assisted reproduction centers. However, the molecular mechanisms underlying early embryonic development arrest remain largely unknown. The objective of this study is to investigate potential candidate hub genes and key signaling pathways involved in early stages of embryonic development. RNA-seq analysis was performed on normal and arrest embryos to study the changes of gene expression during early embryonic development. A total of 520 genes exhibiting differential expression were identified, with 174 genes being upregulated and 346 genes being downregulated. Upregulated genes show enrichment in biosynthesis, cellular proliferation and differentiation, and epigenetic regulation. While downregulated genes exhibit enrichment in transcriptional activity, epigenetic regulation, cell cycle progression, cellular proliferation and ubiquitination. The STRING (search tool for the retravel of interacting genes/proteins) database was utilized to analyze protein-protein interactions among these genes, aiming to enhance comprehension of the potential role of these differentially expressed genes (DEGs). A total of 22 hub genes (highly connected genes) were identified among the DEGs using Cytoscape software. Of these, ERBB2 and VEGFA were upregulated, while the remaining 20 genes (CCNB1, CCNA2, DICER1, NOTCH1, UBE2B, UBE2N, PRMT5, UBE2D1, MAPK3, SOX9, UBE2C, UB2D2, EGF, ACTB, UBA52, SHH, KRAS, UBE2E1, ADAM17 and BRCA2) were downregulated. These hub genes are associated with crucial biological processes such as ubiquitination, cellular senescence, cell proliferation and differentiation, and cell cycle. Among these hub genes, CCNA2 and CCNB1 may be involved in controlling cell cycle, which are critical process in early embryonic development.

Characterization of the long noncoding RNA transcriptome in human preimplantation embryo development.

Infertility remains a human health burden globally. Only a fraction of embryos produced via assisted reproductive technologies (ARTs) develop to the blastocyst stage in vitro. lncRNA abundance changes significantly during human early embryonic development, indicating vital regulatory roles of lncRNAs in this process. The aim of this study is to obtain insights into the transcriptional basis of developmental events. scRNA-seq data and SUPeR-seq data were used to investigate the lncRNA profiles of human preimplantation embryos. The top 50 highly expressed unique and shared lncRNAs in each stage of preimplantation development were identified. Comparative analysis of the two datasets was used to verify the consistent expression patterns of the lncRNAs. Differentially expressed lncRNAs were identified and subjected to functional enrichment analysis. The lncRNA profiles of human preimplantation embryos in the E-MTAB-3929 dataset were similar to those in the GSE71318 dataset. The ratios of overlap among the top 50 highly expressed lncRNAs between two pairs of stages (2-cell stage vs. 4-cell stage and 8-cell stage vs. morula) were aberrantly low compared with those between other stages. Each stage of preimplantation development exhibited unique and shared lncRNAs among the top 50 highly expressed lncRNAs. Among the between-group comparisons, the 2-cell stage vs. 4-cell stage showed the highest number of differentially expressed lncRNAs. Functional enrichment analysis revealed that differentially expressed lncRNAs and their associated super enhancers and RNA binding proteins (RBPs) are closely involved in regulating embryonic development. These lncRNAs could function as important cell markers for distinguishing fetal germ cells. Our study paves the way for understanding the regulation of developmental events, which might be beneficial for improved reproductive outcomes.

The importance of cytoplasmic strings during early human embryonic development.

Objectives: During human in vitro fertilisation (IVF) treatments, embryologists attempt to select the most viable embryos for embryo transfer (ET). Previously, embryos were evaluated based on light microscopic morphological parameters. However, this is currently accomplished by morphokinetic analysis of time-lapse recordings. This technique provides us the opportunity to observe cytoplasmic strings at the blastocyst stage. The aim of this work was to examine the relationship between the presence of cytoplasmic strings (CS) and the embryo viability in human in vitro fertilised embryos. Study design: Herein, we present an evaluation of the morphokinetic data on the development of embryos obtained during IVF treatments performed at the Division of Assisted Reproduction between December 2020 and March 2021. The dynamics of embryo development, embryo morphology, and morphokinetic scores generated by a time-lapse system were compared between the presence of cytoplasmic strings (CS+) and their absence (CS-) at the blastocyst stage. Results: The development of 208 embryos from 78 patients was examined. Moreover, 81.2% of the embryos had CS in the blastocyst stage; 77% of CS existed in embryos created by conventional IVF, while 86% of CS existed in embryos fertilised by intracytoplasmic sperm injection (ICSI) (p = 0.08). A greater number of CS+ embryos developed into a higher quality blastocyst (52.1% vs. 20.5%, p = 0.02). The morphokinetic score values characterising the development of embryos, such as Known Implantation Data Score (KIDScore) and Intelligent Data Analysis (iDAScore), were higher in CS+ groups (KID: 6.1 ± 2.1 vs. 4.7 ± 2.07; iDA: 8.0 ± 1.9 vs. 6.8 ± 2.3, p < 0.01). The dynamics of the early embryo development were similar between the two groups; however, CS+ embryos reached the blastocyst stage significantly earlier (tB: 103.9h vs. tB: 107.6h; p = 0.001). Conclusion: Based on our results, the number of embryos with cytoplasmic strings was higher than that without cytoplasmic strings, and its presence is not related to the fertilisation method. These embryos reached the blastocyst stage earlier, and their morphokinetic (KIDScore and iDAScore) parameters were better. All these results suggest that the presence of CS indicates higher embryo viability. The examination of this feature may help us make decisions about the embryos with higher implantation potential.

Self-patterning of human stem cells into post-implantation lineages

Investigating human development is a substantial scientific challenge due to the technical and ethical limitations of working with embryonic samples. In the face of these difficulties, stem cells have provided an alternative to experimentally model inaccessible stages of human development in vitro1–13. Here we show that human pluripotent stem cells can be triggered to self-organize into three-dimensional structures that recapitulate some key spatiotemporal events of early human post-implantation embryonic development. Our system reproducibly captures spontaneous differentiation and co-development of embryonic epiblast-like and extra-embryonic hypoblast-like lineages, establishes key signalling hubs with secreted modulators and undergoes symmetry breaking-like events. Single-cell transcriptomics confirms differentiation into diverse cell states of the perigastrulating human embryo14,15 without establishing placental cell types, including signatures of post-implantation epiblast, amniotic ectoderm, primitive streak, mesoderm, early extra-embryonic endoderm, as well as initial yolk sac induction. Collectively, our system captures key features of human embryonic development spanning from Carnegie stage16 4–7, offering a reproducible, tractable and scalable experimental platform to understand the basic cellular and molecular mechanisms that underlie human development, including new opportunities to dissect congenital pathologies with high throughput.

P-121 Identification of differentially expressed genes common to the developmental potential, maternal age, and morphology in human blastocysts

Abstract Study question Is it possible to apply RNA-sequencing to assisted reproductive technology (ART) to predict blastocyst quality? Summary answer We identified 14 commonly identified differentially expressed genes by grouping blastocysts according to three distinct parameters such as developmental potential, maternal age, and Gardner score What is known already In ART, while the selection of a suitable embryo for transfer is critical for a successful pregnancy, neither preimplantation genetic testing nor morphological and chronological evaluation of the embryo can fully guarantee a successful pregnancy. Recently, transcriptional events in early human embryonic development have been analyzed using RNA-sequencing (RNA-seq) and researchers are attempting to apply this information to ART. We have reported that 96 differentially expressed genes (DEGs) were identified using RNA-seq of each inner cell mass (ICM) and trophectoderm (TE) in blastocysts classified according to the developmental potential which correlates with pregnancy rate at the ESHRE 37th annual meeting. Study design, size, duration After retrospectively analyzing 1,890 cases undergoing freeze-thaw blastocyst transfer from March 2018 to December 2020 to examine the correlation between blastocyst developmental potential and pregnancy rate, a total 13 blastocysts cryopreserved between February 2011 and September 2018, then scheduled for disposal and with consented, were subjected to RNA-seq to identify genes associated with pregnancy expectation. RNA-seq data were then examined whether common DEGs could be found when classified by maternal age and Gardner score, respectively. Participants/materials, setting, methods Blastocysts were donated by infertile couples undergoing c-IVF or ICSI cycles at the Yamashita Shonan Yume Clinic with informed consent under ethical approval. TE cells and ICM cells were collected from blastocysts classified by developmental potential and subjected to RNA-seq to identify DEGs. In addition, RNA-seq data were regrouped by maternal age and Gardner score to find common DEGs. DEGs (q-value &amp;lt; 0.01) were identified using the R package “DESeq2” (version 1.32.0). Main results and the role of chance When the RNA-seq data obtained from blastocysts classified according to pregnancy expectation were re-grouped by maternal age and re-analyzed, we identified 7 and 17 genes that were down- and up-regulated in the elder group, respectively, in ICM. In TE, 2 and 12 genes were down- and up-regulated in the elder group, respectively. On the other hand, when re-grouped by Gardner score (&amp;lt;BB versus &amp;lt;BB), we identified 26 and 29 genes that were down- and up-regulated in the poor group, respectively, in ICM. In TE, 64 and 20 genes were down- and up-regulated in the poor group, respectively. A comparison of these genes and DEGs identified by the pregnancy expectation found 14 common genes. By comparison with the elder group, we identified one gene (UCHL1) that was commonly up-regulated in TE. By comparison with the poor Gardner score group, one gene (CMTM7) was commonly down-regulated and three genes (LINC00458, DYNLRB1, and UBL4A) were commonly up-regulated in ICM, and 5 genes (LRIG3, SLC28A3, CXADR, LOC727896, and ATP1B1) were commonly down-regulated and 5 genes (UCHL1, BZW2, NABP1, HEXIM1, and PLEKHA6) were commonly up-regulated in TE. Limitations, reasons for caution Although we established an expected pregnancy rate concerning the degree of blastocyst development from retrospective clinical outcomes and used it as a surrogate marker for assigning biopsied blastocysts to different analysis groups, it remains unknown whether the gene expression profiles accurately reflect the pregnancy outcomes. Wider implications of the findings UCHL1 expression was commonly increased with lower blastocyst developmental potential, higher maternal age, and lower Gardner score. Since UCHL1 has been reported to be essential for blastocyst development in mice, our results suggest that UCHL1 may also be a marker of blastocyst quality in humans. Trial registration number not applicable

P-148 The importance of filopodia during early human embryonic development

Abstract Study question The aim of this study was to examine the relation between the presence of filopodia and the embryo viability in human in vitro fertilised embryos. Summary answer The presence of filopodia related to a faster blastocyst development, a better blastocyst quality and higher morphokinetic scores which indicates a higher embryo viability. What is known already Filopodia are involved in numerous cellular processes, for example cell migration, neuronal extension growth and during embryonic development. There is no uniform name to describe these cytoplasmic string structures in the literature. Beside the frequent presence in different cell functions, we have limited information about its function in human embryos. In the early 2000s, the occurrence of filopodia or cytoplasmic strings at the blastocyst stage was classified as a negative sign of embryo viability, but more recently, it has been described as a positive feature, however, the importance and clinical significance of this structure is still controversial. Study design, size, duration Evaluation of the morphokinetic data on the development of 208 embryos from 78 IVF cycles. Examination was carried out at the Division of Assisted Reproduction, Department of Obstetrics and Gynaecology, Semmelweis University, Budapest between December 2020 and March 2021. Participants/materials, setting, methods Embryos from both conventional IVF (cIVF) and intracytoplasmic sperm injection (ICSI) treatments were analysed and evaluated. The dynamics of embryo development, embryo morphology and morphokinetic scores generated by time-lapse system was compared between the embryos with filopodia (FP+) and embryos without filopodia- (FP-) groups. Main results and the role of chance 81.2% of the embryos had filopodia in blastocyst stage. Embryos created by cIVF had 77%, while embryos fertilised by ICSI had 86% of filopodia presence (p = 0.08). FP+ embryos developed in a greater number into a higher quality blastocyst (52.1% vs 20.5%, p = 0,02), their KIDScore was higher (6.1 ± 2.1 vs 4,7 ± 2,07, p &amp;lt; 0.001) and they showed a tendency of higher implantation rate (39.7% vs 14.3% p = 0.16) than the FP- group. The dynamic of the early embryo development was similar between the two groups, however, FP+ embryos reached blastocyst stage significantly earlier (tB: 103.9 hours vs. tB: 107.6 hours; p = 0.007). Based on our results, there was a higher number of embryos with filopodia than without, and the presence of it is not related to the fertilisation method. These embryos developed faster into a blastocyst stage, their morphokinetic parameters were better. KIDScore and iDAScore embryo evaluation systems also recommend in a greater number the FP+ blastocysts for ET. We could also observe vesicle like transport along the filopodia, which leads us to believe that there might be a molecular communication between the differentiating cell populations. Limitations, reasons for caution Due to the low number of cases, the higher implantation rate is not significant. Wider implications of the findings The examination of this feature improves our knowledge of the early embryonic development and may help us to make decisions about the embryos with higher implantation potential. Trial registration number Not applicable

Morphokinetic analysis of early human embryonic development and its relationship to endometriosis resection: a retrospective time-lapse study using the KIDScore™ D3 and D5 implantation data algorithm

Research questionDoes complete resection of endometriosis improve embryo quality as assessed by morphokinetic parameters using time-lapse microscopy?DesignFor this retrospective study we analysed 237 fertilised, cultured and transferred embryos from 128 fresh IVF and/ or ICSI transfer cycles. Endometriosis was confirmed or excluded by laparoscopy. Patients were stimulated with recombinant FSH using GnRH agonist and antagonist protocols. After fertilisation, a time-lapse incubation system was used for observation. Embryo quality was assessed using the KIDScore™ D3 and D5 implantation data algorithm.ResultsThe analysis showed a median KIDScore™ D5 of 2.6 (on a scale of 1 to 9.9) for embryos from patients with endometriosis without complete resection. The control group without endometriosis achieved a score of 6.8 (p = 0.003). The median score for embryos from endometriosis patients with complete resection was 7.2, which was a significant increase compared to embryos from patients without complete resection (p = 0.002). We observed an effect size of r = 0.4 for complete resection versus no resection of endometriosis using the KIDScore™ D5. There were no differences in KIDScore™ D3 between the three patient groups. Pregnancy and miscarriage rates showed the same clinical trends. In three of our four case series of patients who underwent IVF/ ICSI cycles before and after complete resection, we found a marked improvement in embryo quality after complete resection.ConclusionsComplete resection of endometriosis could significantly improve the otherwise poor embryo quality of patients undergoing IVF-procedures. The data, therefore, strongly support recommending surgery to patients with endometriosis prior to assisted reproduction.

Metabolomics Identifies a Panel of Diagnostic Biomarkers for Early Human Embryonic Development Arrest.

Early embryonic development arrest (EEDA) is a unique form of early spontaneous abortion in pregnant women, which is previously suggested to be associated with metabolic abnormalities. Noninvasive biomarkers would significantly improve its diagnosis and clinical outcome. Here, we performed a targeted metabolomics study in plasma from EEDA patients (n = 27) and normal pregnant women (NPW, n = 27) using liquid chromatography coupled with mass spectrometry (LC-MS) to identify potential diagnostic marker metabolites. Our results showed significantly different plasma metabolic profiles between EEDA patients and NPW. Particularly, EEDA patients showed significant alterations in amino acid, carbohydrate, and vitamin metabolism, which were characterized by 21 significantly increased metabolites and five decreased metabolites in plasma. Further receiver operating characteristic analysis showed that an optimal combination of S-methyl-5'-thioadenosine, kynurenine, leucine, and malate could be used as a panel of metabolites for EEDA diagnosis. The area under the curve of the metabolite panel was 0.941, suggesting a better performance than any single metabolite for the diagnosis of EEDA. In summary, our study identifies a panel of differential metabolites in plasma that could act as potential biomarkers for the diagnosis of EEDA in clinical settings.

The Prognostic Significance of FOXD1 Expression in Head and Neck Squamous Cell Carcinoma

It has been reported that forkhead box D1 (FOXD1) plays an established role in human early embryonic development and is broadly involved in various malignancies. However, there is limited information regarding FOXD1 expression in head and neck squamous cell carcinoma (HNSCC). This present study aimed to explore the clinical significance of FOXD1 in patients with HNSCC. Tissue microarrays of 334 primary HNSCC patients who underwent surgery between 2008 and 2010 at Sun Yat-sen University Cancer Center were investigated by immunohistochemistry regarding FOXD1 expression. χ2 test was used to estimate the relationship of FOXD1 expression with clinicopathologic characteristics. Univariate and multivariate analyses were performed to identify FOXD1 expression as an independent prognostic indicator of overall survival (OS) and disease-free survival (DFS). FOXD1 expression is closely associated with postoperative recurrence. HNSCC patients with high FOXD1 expression have poorer prognoses than the low-expression group (p < 0.05). According to multivariate analysis, FOXD1 was an independent prognostic factor for OS and DFS. The results revealed that FOXD1 could be a prognostic factor for HNSCC and might serve as a potential target for novel therapies.

Developmental toxicity assessment of neonicotinoids and organophosphate esters with a human embryonic stem cell- and metabolism-based fast-screening model

In recent years, neonicotinoids (NEOs) and organophosphate esters (OPEs) have been widely used as substitutes for traditional pesticides and brominated flame-retardants, respectively. Previous studies have shown that those compounds can be frequently detected in environmental and human samples, are able to penetrate the placental barrier, and are toxic to animals. Thus, it is reasonable to speculate that NEOs and OPEs may have potential adverse effects in humans, especially during development. We employed a human embryonic stem cell differentiation- and liver S9 fraction metabolism-based fast screening model to assess the potential embryonic toxicity of those two types of chemicals. We show that four NEO and five OPE prototypes targeted mostly ectoderm specification, as neural ectoderm and neural crest genes were down-regulated, and surface ectoderm and placode markers up-regulated. Human liver S9 fraction's treatment could generally reduce the effects of the chemicals, except in a few specific instances, indicating the liver may detoxify NEOs and OPEs. Our findings suggest that NEOs and OPEs interfere with human early embryonic development.

Naive Pluripotent and Trophoblastic Stem Cell Lines as a Model for Detecting Missing Proteins in the Context of the Chromosome-Centric Human Proteome Project.

The Chromosome-centric Human Proteome Project (C-HPP) aims at identifying the proteins as gene products encoded by the human genome, characterizing their isoforms and functions. The existence of products has now been confirmed for 93.2% of the genes at the protein level. The remaining mostly correspond to proteins of low abundance or difficult to access. Over the past years, we have significantly contributed to the identification of missing proteins in the human spermatozoa. We pursue our search in the reproductive sphere with a focus on early human embryonic development. Pluripotent cells, developing into the fetus, and trophoblast cells, giving rise to the placenta, emerge during the first weeks. This emergence is a focus of scientists working in the field of reproduction, placentation and regenerative medicine. Most knowledge has been harnessed by transcriptomic analysis. Interestingly, some genes are uniquely expressed in those cells, giving the opportunity to uncover new proteins that might play a crucial role in setting up the molecular events underlying early embryonic development. Here, we analyzed naive pluripotent and trophoblastic stem cells and discovered 4 new missing proteins, thus contributing to the C-HPP. The mass spectrometry proteomics data was deposited on ProteomeXchange under the data set identifier PXD035768.

Analysis of Influenza A virus infection in human induced pluripotent stem cells (hiPSCs) and their derivatives

Influenza A virus (IAV) infection in pregnant women is a major public health concern. However, the effect of IAV infection on human embryogenesis is still unclear. Here we show that human induced pluripotent stem cells (hiPSCs) and hiPSC-derived ectodermal, mesodermal and endodermal cells are susceptible to IAV infection. These cell types stained positive for α2,6-linked sialic acid, the receptor for IAV infection expressed on the cell surface. While hiPSCs produced high viral titers for up to 7 days with increasing infected cell number suggesting that the viral progenies produced from hiPSCs without exogenous protease were infectious and could spread to other cells, the three germ-layer cells showed a decline in viral titers suggesting the lack of viral spreading. Amongst the three germ layers, endodermal cells were less susceptible than ectodermal and mesodermal cells. These results indicate the permissiveness of cells of early embryogenesis, and suggest a risk of detrimental effects of IAV infection in early human embryonic development.