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Understanding the molecular circuitries that govern early embryogenesis is important, yet our knowledge of these in human preimplantation development remains limited. Small non-coding RNAs (sncRNAs) can regulate gene expression and thus impact blastocyst formation, however, the expression of specific biotypes and their dynamics during preimplantation development remains unknown. Here we identify the abundance of and kinetics of piRNA, rRNA, snoRNA, tRNA, and miRNA from embryonic day (E)3-7 and isolate specific miRNAs and snoRNAs of particular importance in blastocyst formation and pluripotency. These sncRNAs correspond to specific genomic hotspots: an enrichment of the chromosome 19 miRNA cluster (C19MC) in the trophectoderm (TE), and the chromosome 14 miRNA cluster (C14MC) and MEG8-related snoRNAs in the inner cell mass (ICM), which may serve as 'master regulators' of potency and lineage. Additionally, we observe a developmental transition with 21 isomiRs and in tRNA fragment (tRF) codon usage and identify two novel miRNAs. Our analysis provides a comprehensive measure of sncRNA biotypes and their corresponding dynamics throughout human preimplantation development, providing an extensive resource. Better understanding the sncRNA regulatory programmes in human embryogenesis will inform strategies to improve embryo development and outcomes of assisted reproductive technologies. We anticipate broad usage of our data as a resource for studies aimed at understanding embryogenesis, optimising stem cell-based models, assisted reproductive technology, and stem cell biology., (© 2024. The Author(s).)

Single-cell genomics allow the characterization and quantification of molecular heterogeneity from a wide variety of tissues. Here, we describe the manual dissociation and collection of single cells, a method adapted for the characterization of precious small tissues like preimplantation embryos. We also describe the acquisition of mouse embryos by flushing of the oviducts. The cells can then be used in multiple sequencing protocols, for example, Smart-seq2, Smart-seq3, smallseq, and scBSseq., (© 2023. Springer Science+Business Media, LLC.)

Fluorescence in situ hybridization (FISH) provides a valuable tool for studying the spatial localization of and expression level of genes and cell function in diverse biological contexts. In this chapter, we describe a protocol for the simultaneous detection of RNA (including single-molecule (sm)RNA) and DNA in mammalian embryos using FISH. RNA FISH is a technique that enables the detection and visualization of specific RNA molecules within cells. With advancements in technology, the sensitivity and specificity of RNA FISH has been improved to allow the detection of individual mRNA molecules. Both RNA and smRNA are detected using a set of fluorescent-labeled probes, which are complementary to a specific nucleotide sequence corresponding to the gene of interest. These probes hybridize to the target RNA molecules, enabling the simultaneous detection of multiple RNAs within the same cell or tissue. DNA FISH is performed using probes directed at the DNA sequence to detect the genome region of interest. In this chapter, we provide a protocol to process mammalian embryos for FISH with probe examples specifically for studying X-Chromosome activity. By utilizing other probe designs, this protocol can be adapted for the visualization and quantification of other genes and chromosomal regions of interest., (© 2023. Springer Science+Business Media, LLC.)

The development of single-cell multiomics has provided the ability to systematically investigate cellular diversity and heterogeneity in different biological systems via comprehensive delineations of individual cellular states. Single-cell RNA sequencing in particular has served as a powerful tool to the study of the molecular circuitries underlying preimplantation embryonic development in both the mouse and human. Here we describe a method to elucidate the cellular dynamics of the embryo further by performing both single-cell RNA sequencing (Smart-Seq2) and single-cell small non-coding RNA sequencing (Small-Seq) on the same individual embryonic cell., (© 2023. Springer Science+Business Media, LLC.)

Studying various animal models is important for comparative biology and to better understand evolutionary development. Furthermore, when aiming to translate findings to human development, it is crucial to select an appropriate animal model that closely resembles the specific aspect of development under study. The guinea pig is highlighted as a useful platform for reproductive studies due to similarities in in utero development and general physiology with the human. This chapter outlines the methods required for guinea pig mating and collection of embryos for in vitro culture and molecular characterization. Specifically, this chapter provides detailed guidance on monitoring the estrus cycle to determine the mating time, performing a vaginal flush and smear to confirm successful mating, performing euthanasia of the guinea pig, and flushing in vivo embryos. Once collected, the embryos can be utilized for numerous downstream applications. Here we will cover embryo culturing and processing embryos for immunofluorescence., (© 2023. Springer Science+Business Media, LLC.)

Proper formation of the blastocyst, including the specification of the first embryonic cellular lineages, is required to ensure healthy embryo development and can significantly impact the success of assisted reproductive technologies (ARTs). However, the regulatory role of microRNAs in early development, particularly in the context of preimplantation lineage specification, remains largely unknown. Taking a cross-species approach, this review aims to summarize the expression dynamics and functional significance of microRNAs in the differentiation and maintenance of lineage identity in both the mouse and the human. Findings are consolidated from studies conducted using in vitro embryonic stem cell models representing the epiblast, trophectoderm, and primitive endoderm lineages (modeled by naïve embryonic stem cells, trophoblast stem cells, and extraembryonic endoderm stem cells, respectively) to provide insight on what may be occurring in the embryo. Additionally, studies directly conducted in both mouse and human embryos are discussed, emphasizing similarities to the stem cell models and the gaps in our understanding, which will hopefully lead to further investigation of these areas. By unraveling the intricate mechanisms by which microRNAs regulate the specification and maintenance of cellular lineages in the blastocyst, we can leverage this knowledge to further optimize stem cell-based models such as the blastoids, enhance embryo competence, and develop methods of non-invasive embryo selection, which can potentially increase the success rates of assisted reproductive technologies and improve the experiences of those receiving fertility treatments., (Copyright ©2023, Yale Journal of Biology and Medicine.)

In Brief: Human embryogenesis still remains largely unexplored. This review helps identify some of our current gaps in knowledge pertaining to preimplantation development, which may have implications for understanding fundamental aspects of human development, assisted reproductive technologies, and stem cell biology., Abstract: Preimplantation development is arguably one of the most critical stages of embryogenesis. Beginning with the formation of the totipotent zygote post-fertilization, a series of cell divisions, and a complex coordination of physical cues, molecular signals and changes in gene expression lead to the formation of the blastocyst, a structure capable of implanting into the uterine wall. The blastocyst is composed of more specified cellular lineages, which will give rise to every tissue of the developing organism as well as the extra-embryonic lineages which support fetal growth. While the mouse has been used as a model to understand the events of preimplantation development for decades, in recent years, an expanding body of work has been conducted using the human embryo. These studies have identified some crucial species differences, particularly in the transcriptional and spatio-temporal expression of lineage markers and responses to cell signaling perturbations. This review compares recent findings on preimplantation development in mouse and human, with a focus on the specification of the first cellular lineages. Highlighting differences and noting mechanisms that require further examination in the human embryo is of critical importance for both the accurate translation of results from the mouse model and our overall understanding of mammalian development. We further highlight the latest advancement in reproductive research, the development of the 3D stem cell-based models known as 'blastoids'. The knowledge discussed in this review has major clinical implications for assisted reproductive technologies such as in vitro fertilization and for applications in stem cell biology.

The preconceptual, intrauterine, and early life environments can have a profound and long-lasting impact on the developmental trajectories and health outcomes of the offspring. Given the relatively low success rates of assisted reproductive technologies (ART; ∼25%), additives and adjuvants, such as glucocorticoids, are used to improve the success rate. Considering the dynamic developmental events that occur during this window, these exposures may alter blastocyst formation at a molecular level, and as such, affect not only the viability of the embryo and the ability of the blastocyst to implant, but also the developmental trajectory of the first three cell lineages, ultimately influencing the physiology of the embryo. In this study, we present a comprehensive single-cell transcriptome, methylome, and small RNA atlas in the day 7 human embryo. We show that, despite no change in morphology and developmental features, preimplantation glucocorticoid exposure reprograms the molecular profile of the trophectoderm (TE) lineage, and these changes are associated with an altered metabolic and inflammatory response. Our data also suggest that glucocorticoids can precociously mature the TE sublineages, supported by the presence of extravillous trophoblast markers in the polar sublineage and presence of X Chromosome dosage compensation. Further, we have elucidated that epigenetic regulation-DNA methylation and microRNAs (miRNAs)-likely underlies the transcriptional changes observed. This study suggests that exposures to exogenous compounds during preimplantation may unintentionally reprogram the human embryo, possibly leading to suboptimal development and longer-term health outcomes., (© 2022 Zhao et al.; Published by Cold Spring Harbor Laboratory Press.)