Classification of Atretic Small Antral Follicles in the Human Ovary.

The reproductive lifespan in humans is regulated by a delicate cyclical balance between follicular recruitment and atresia in the ovary. The majority of the small antral follicles present in the ovary are progressively lost through atresia without reaching dominance, but this process remains largely underexplored. In our study, we investigated the characteristics of atretic small antral follicles and proposed a classification system based on molecular changes observed in granulosa cells, theca cells, and extracellular matrix deposition. Our findings revealed that atresia spreads in the follicle with wave-like dynamics, initiating away from the cumulus granulosa cells. We also observed an enrichment of CD68+ macrophages in the antrum during the progression of follicular atresia. This work not only provides criteria for classifying three stages of follicular atresia in small antral follicles in the human ovary but also serves as a foundation for understanding follicular degeneration and ultimately preventing or treating premature ovarian failure. Understanding follicular remodeling in the ovary could provide a means to increase the number of usable follicles and delay the depletion of the follicular reserve, increasing the reproductive lifespan.

Teratoma Assay for Testing Pluripotency and Malignancy of Stem Cells: Insufficient Reporting and Uptake of Animal-Free Methods-A Systematic Review.

Pluripotency describes the ability of stem cells to differentiate into derivatives of the three germ layers. In reporting new human pluripotent stem cell lines, their clonal derivatives or the safety of differentiated derivatives for transplantation, assessment of pluripotency is essential. Historically, the ability to form teratomas in vivo containing different somatic cell types following injection into immunodeficient mice has been regarded as functional evidence of pluripotency. In addition, the teratomas formed can be analyzed for the presence of malignant cells. However, use of this assay has been subject to scrutiny for ethical reasons on animal use and due to the lack of standardization in how it is used, therefore questioning its accuracy. In vitro alternatives for assessing pluripotency have been developed such as ScoreCard and PluriTest. However, it is unknown whether this has resulted in reduced use of the teratoma assay. Here, we systematically reviewed how the teratoma assay was reported in publications between 1998 (when the first human embryonic stem cell line was described) and 2021. Our analysis of >400 publications showed that in contrast to expectations, reporting of the teratoma assay has not improved: methods are not yet standardized, and malignancy was examined in only a relatively small percentage of assays. In addition, its use has not decreased since the implementation of the ARRIVE guidelines on reduction of animal use (2010) or the introduction of ScoreCard (2015) and PluriTest (2011). The teratoma assay is still the preferred method to assess the presence of undifferentiated cells in a differentiated cell product for transplantation since the in vitro assays alone are not generally accepted by the regulatory authorities for safety assessment. This highlights the remaining need for an in vitro assay to test malignancy of stem cells.

Stay on the road: from germ cell specification to gonadal colonization in mammals.

The founder cells of the gametes are primordial germ cells (PGCs). In mammals, PGCs are specified early during embryonic development, at the boundary between embryonic and extraembryonic tissue, long before their later residences, the gonads, have developed. Despite the differences in form and behaviour when differentiated into oocytes or sperm cells, in the period between specification and gonadal colonization, male and female PGCs are morphologically indistinct and largely regulated by similar mechanisms. Here, we compare different modes and mechanisms that lead to the formation of PGCs, putting in context protocols that are in place to differentiate both human and mouse pluripotent stem cells into PGC-like cells. In addition, we review important aspects of the migration of PGCs to the gonadal ridges, where they undergo further sex-specific differentiation. Defects in migration need to be effectively corrected, as misplaced PGCs can become tumorigenic. Concluding, a combination of in vivo studies and the development of adequate innovative in vitro models, ensuring both robustness and standardization, are providing us with the tools for a greater understanding of the first steps of gametogenesis and to develop disease models to study the origin of germ cell tumours. This article is part of the theme issue 'Extraembryonic tissues: exploring concepts, definitions and functions across the animal kingdom'.

The development of the amnion in mice and other amniotes.

The amnion is an extraembryonic tissue that evolutionarily allowed embryos of all amniotes to develop in a transient and local aquatic environment. Despite the importance of this tissue, very little is known about its formation and its molecular characteristics. In this review, we have compared the basic organization of the extraembryonic membranes in amniotes and describe the two types of amniogenesis, folding and cavitation. We then zoom in on the atypical development of the amnion in mice that occurs via the formation of a single posterior amniochorionic fold. Moreover, we consolidate lineage tracing data to better understand the spatial and temporal origin of the progenitors of amniotic ectoderm, and visualize the behaviour of their descendants in the extraembryonic-embryonic junctional region. This analysis provides new insight on amnion development and expansion. Finally, using an online-available dataset of single-cell transcriptomics during the gastrulation period in mice, we provide bioinformatic analysis of the molecular signature of amniotic ectoderm and amniotic mesoderm. The amnion is a tissue with unique biomechanical properties that deserves to be better understood. This article is part of the theme issue 'Extraembryonic tissues: exploring concepts, definitions and functions across the animal kingdom'.

The centriolar satellite protein Cfap53 facilitates formation of the zygotic microtubule organizing center in the zebrafish embryo.

In embryos of most animal species, the zygotic centrosome is assembled by the centriole derived from the sperm cell and pericentriolar proteins present in the oocyte. This zygotic centrosome acts as a microtubule organizing center (MTOC) to assemble the sperm aster and mitotic spindle. As MTOC formation has been studied mainly in adult cells, very little is known about the formation of the zygotic MTOC. Here, we show that zebrafish (Danio rerio) embryos lacking either maternal or paternal Cfap53, a centriolar satellite protein, arrest during the first cell cycle. Although Cfap53 is dispensable for sperm aster function, it aids proper formation of the mitotic spindle. During cell division, Cfap53 colocalizes with γ-tubulin and with other centrosomal and centriolar satellite proteins at the MTOC. Furthermore, we find that γ-tubulin localization at the MTOC is impaired in the absence of Cfap53. Based on these results, we propose a model in which Cfap53 deposited in the oocyte and the sperm participates in the organization of the zygotic MTOC to allow mitotic spindle formation.

Neural correlates of thematic role assignment for passives in Standard Indonesian.

Previous studies of multiple languages have found processing differences between patient-first and agent-first word orders. However, the results are inconsistent as they do not identify a specific ERP component as a unique correlate of thematic role processing. Furthermore, these studies generally confound word order with frequency, as patient-first structures tend to be infrequent in the languages that have been investigated. There is evidence that frequency at the sentence level plays a significant role in language processing. To address this potential confounding variable, we will test a language where the non-canonical sentences are more frequent and are comparable to the canonical sentences, namely Standard Indonesian. In this language, there is evidence from acquisition, corpus, and clinical data indicates that the use of passive is frequent and salient. One instance of this difference can be demonstrated by the fact that it has been suggested that frequency may be the reason why Indonesian-speaking aphasic speakers do not have impairments in the comprehension of passives, whereas speakers of other languages with aphasia often do. In the present study, we will test 50 native speakers of Indonesian using 100 sentences (50 active and 50 passive sentences). If the neural correlates of thematic role processing are not observed in the critical region of the sentence (the prefix of the verb), this would suggest that the previous results were indeed influenced by frequency, but if we find that specific ERPs are connected to the hypothesized syntactic operations, this would further reinforce the existing evidence of the increased cognitive load required to process more syntactically complicated sentences.

Genetic Basis of Dilated Cardiomyopathy in Dogs and Its Potential as a Bidirectional Model.

Cardiac disease is a leading cause of death for both humans and dogs. Genetic cardiomyopathies, including dilated cardiomyopathy (DCM), account for a proportion of these cases in both species. Patients may suffer from ventricular enlargement and systolic dysfunction resulting in congestive heart failure and ventricular arrhythmias with high risk for sudden cardiac death. Although canine DCM has similar disease progression and subtypes as in humans, only a few candidate genes have been found to be associated with DCM while the genetic background of human DCM has been more thoroughly studied. Additionally, experimental disease models using induced pluripotent stem cells have been widely adopted in the study of human genetic cardiomyopathy but have not yet been fully adapted for the in-depth study of canine genetic cardiomyopathies. The clinical presentation of DCM is extremely heterogeneous for both species with differences occurring based on sex predisposition, age of onset, and the rate of disease progression. Both genetic predisposition and environmental factors play a role in disease development which are identical in dogs and humans in contrast to other experimental animals. Interestingly, different dog breeds have been shown to develop distinct DCM phenotypes, and this presents a unique opportunity for modeling as there are multiple breed-specific models for DCM with less genetic variance than human DCM. A better understanding of DCM in dogs has the potential for improved selection for breeding and could lead to better overall care and treatment for human and canine DCM patients. At the same time, progress in research made for human DCM can have a positive impact on the care given to dogs affected by DCM. Therefore, this review will analyze the feasibility of canines as a naturally occurring bidirectional disease model for DCM in both species. The histopathology of the myocardium in canine DCM will be evaluated in three different breeds compared to control tissue, and the known genetics that contributes to both canine and human DCM will be summarized. Lastly, the prospect of canine iPSCs as a novel method to uncover the contributions of genetic variants to the pathogenesis of canine DCM will be introduced along with the applications for disease modeling and treatment.

Silencing XIST on the future active X: Searching human and bovine preimplantation embryos for the repressor.

X inactivation is the means of equalizing the dosage of X chromosomal genes in male and female eutherian mammals, so that only one X is active in each cell. The XIST locus (in cis) on each additional X chromosome initiates the transcriptional silence of that chromosome, making it an inactive X. How the active X in both males and females is protected from inactivation by its own XIST locus is not well understood in any mammal. Previous studies of autosomal duplications suggest that gene(s) on the short arm of human chromosome 19 repress XIST on the active X. Here, we examine the time of transcription of some candidate genes in preimplantation embryos using single-cell RNA sequencing data from human embryos and qRT-PCR from bovine embryos. The candidate genes assayed are those transcribed from 19p13.3-13.2, which are widely expressed and can remodel chromatin. Our results confirm that XIST is expressed at low levels from the future active X in embryos of both sexes; they also show that the XIST locus is repressed in both sexes when pluripotency factors are being upregulated, during the 4-8 cell and morula stages in human and bovine embryos - well before the early blastocyst (E5) when XIST on the inactive X in females starts to be upregulated. Our data suggest a role for DNMT1, UHRF1, SAFB and SAFB2 in XIST repression; they also exclude XACT and other 19p candidate genes and provide the transcriptional timing for some genes not previously assayed in human or bovine preimplantation embryos.

Humanised Mice and Immunodeficient Mice (NSG) Are Equally Sensitive for Prediction of Stem Cell Malignancy in the Teratoma Assay.

The use of human pluripotent stem cells (hPSCs) in regenerative medicine has great potential. However, it is important to exclude that these cells can undergo malignant transformation, which could lead to the development of malignant tumours. This property of hPSCs is currently being tested using the teratoma assay, through which cells are injected into immunodeficient mice. Transplantation of stem cells in immunocompromised recipient animals certainly has a much higher incidence of tumour formation. On the other hand, the results obtained in immunodeficient mice could indicate a risk of tumour formation that is practically not present in the human immunocompetent recipient. The presence of a humanised immune system might be more representative of the human situation; therefore, we investigated if the demonstrated malignant features of chosen and well-characterised stem cell lines could be retrieved and if new features could arise in a humanised mouse model. Hu-CD34NSGTM (HIS) mice were compared side by side with immunocompromised mice (NSG) after injection of a set of benign (LU07) and malignant (LU07+dox and 2102Ep) cell lines. Analysis of the tumour development, histological composition, pathology evaluation, and malignancy-associated miRNA expression levels, both in tumour and plasma samples, revealed no differences among mouse groups. This indicates that the HIS mouse model is comparable to, but not more sensitive than, the NSG immunodeficient model for studying the malignancy of stem cells. Since in vivo teratoma assay is cumbersome, in vitro methods for the detection of malignancy are urgently needed.

Lysophosphatidic Acid Accelerates Bovine In Vitro-Produced Blastocyst Formation through the Hippo/YAP Pathway.

The segregation of trophectoderm (TE) and inner cell mass in early embryos is driven primarily by the transcription factor CDX2. The signals that trigger CDX2 activation are, however, less clear. In mouse embryos, the Hippo-YAP signaling pathway is important for the activation of CDX2 expression; it is less clear whether this relationship is conserved in other mammals. Lysophosphatidic acid (LPA) has been reported to increase YAP levels by inhibiting its degradation. In this study, we cultured bovine embryos in the presence of LPA and examined changes in gene and protein expression. LPA was found to accelerate the onset of blastocyst formation on days 5 and 6, without changing the TE/inner cell mass ratio. We further observed that the expression of TAZ and TEAD4 was up-regulated, and YAP was overexpressed, in LPA-treated day 6 embryos. However, LPA-induced up-regulation of CDX2 expression was only evident in day 8 embryos. Overall, our data suggest that the Hippo signaling pathway is involved in the initiation of bovine blastocyst formation, but does not affect the cell lineage constitution of blastocysts.

Reverse transcription priming methods affect normalisation choices for gene expression levels in oocytes and early embryos.

Mammalian oocytes and embryos rely exclusively on maternal mRNAs to accomplish early developmental processes. Since oocytes and early embryos are transcriptionally silent after meiotic resumption, most of the synthesised maternal mRNA does not undergo immediate translation but is instead stored in the oocyte. Quantitative RT-PCR is commonly used to quantify mRNA levels, and correct quantification relies on reverse transcription and the choice of reference genes. Different methods for reverse transcription may affect gene expression determination in oocytes. In this study, we examined the suitability of either random or oligo(dT) primers for reverse transcription to be used for quantitative RT-PCR. We further looked for changes in poly(A) length of the maternal mRNAs during oocyte maturation. Our data indicate that depending on the method of reverse transcription, the optimal combination of reference genes for normalisation differed. Surprisingly, we observed a shortening of the poly(A) tail lengths of maternal mRNA as oocytes progressed from germinal vesicle to metaphase II. Overall, our findings suggest dynamic maternal regulation of mRNA structure and gene expression during oocyte maturation and early embryo development.

Susceptibility of Oocytes from Gilts and Sows to Beauvericin and Deoxynivalenol and Its Relationship with Oxidative Stress.

Beauvericin (BEA) and deoxynivalenol are toxins produced by Fusarium species that can contaminate food and feed. The aim of this study was to assess the effects of these mycotoxins on the maturation of oocytes from gilts and sows. Furthermore, the antioxidant profiles in the oocytes' environment were assessed. Cumulus-oocyte-complexes (COCs) from gilts and sows were exposed to beauvericin (BEA) or deoxynivalenol (DON) and matured in vitro. As an extra control, these COCs were also exposed to reactive oxygen species (ROS). The maturation was mostly impaired when oocytes from gilts were exposed to 0.02 µmol/L DON. Oocytes from sows were able to mature even in the presence of 5 µmol/L BEA. However, the maturation rate of gilt oocytes was already impaired by 0.5 µmol/L BEA. It was observed that superoxide dismutase (SOD) and glutathione (GSH) levels in the follicular fluid (FF) of gilt oocytes was higher than that from sows. However, the expression of SOD1 and glutathione synthetase (GSS) was higher in the oocytes from sows than in those from gilts. Although DON and BEA impair cell development by diverse mechanisms, this redox imbalance may partially explain the vulnerability of gilt oocytes to these mycotoxins.

Beauvericin alters the expression of genes coding for key proteins of the mitochondrial chain in ovine cumulus-oocyte complexes.

Beauvericin (BEA) is a member of the enniatin family of mycotoxins which has received increasing interest because of frequent occurrence in food and feed. By its ionophoric properties, BEA is able to alter membrane ion permeability uncoupling oxidative phosphorylation. It was also shown to alter oocyte mitochondrial function. In this study, the effects of BEA at 0.5, 1, ,3 and 5 µmol/L on expression of genes coding for key proteins of the mitochondrial chain in ovine oocytes and cumulus cells were evaluated at different time points of in vitro maturation (IVM), germinal vesicle (GV; t = 0), metaphase I (MI; t = 7 h), and metaphase II (MII; t = 24 h). The expression of nuclear (TFAM, NDUFA12, UQCRH, COX4, ATP5O) and mitochondrial (ND1, COX1, COX2, ATP6, ATP8) genes coding for proteins of Complexes I, III, IV, and V was analyzed by qRT-PCR. After BEA exposure, perturbed expression of all genes was observed in cumulus cells and in oocytes at the MI stage (7 h IVM). Expression of ND1, UQCRH, COX4 and ATP5O was downregulated in cumulus cells and upregulated in oocytes starting from 0.5 µmol/L BEA. Expression of TFAM, NDUFA12, COX1, COX2, ATP6, and ATP8 was upregulated starting from 1 µmol/L in cumulus cells and from 3 µmol/L in oocytes. Cumulus cells and oocytes displayed different gene expression patterns upon BEA exposure. The downregulation in cumulus cells of four genes coding for proteins of mitochondrial complexes could represent a major toxic event induced by BEA on the cumulus-oocyte complex which may result in mitochondrial functional alteration.

One-step automated bioprinting-based method for cumulus-oocyte complex microencapsulation for 3D in vitro maturation.

Three-dimensional in vitro maturation (3D IVM) is a promising approach to improve IVM efficiency as it could prevent cumulus-oocyte complex (COC) flattening and preserve its structural and functional integrity. Methods reported to date have low reproducibility and validation studies are limited. In this study, a bioprinting based production process for generating microbeads containing a COC (COC-microbeads) was optimized and its validity tested in a large animal model (sheep). Alginate microbeads were produced and characterized for size, shape and stability under culture conditions. COC encapsulation had high efficiency and reproducibility and cumulus integrity was preserved. COC-microbeads underwent IVM, with COCs cultured in standard 2D IVM as controls. After IVM, oocytes were analyzed for nuclear chromatin configuration, bioenergetic/oxidative status and transcriptional activity of genes biomarker of mitochondrial activity (TFAM, ATP6, ATP8) and oocyte developmental competence (KHDC3, NLRP5, OOEP and TLE6). The 3D system supported oocyte nuclear maturation more efficiently than the 2D control (P<0.05). Ooplasmic mitochondrial activity and reactive oxygen species (ROS) generation ability were increased (P<0.05). Up-regulation of TFAM, ATP6 and ATP8 and down-regulation of KHDC3, NLRP5 expression were observed in 3D IVM. In conclusion, the new bioprinting method for producing COC-microbeads has high reproducibility and efficiency. Moreover, 3D IVM improves oocyte nuclear maturation and relevant parameters of oocyte cytoplasmic maturation and could be used for clinical and toxicological applications.

Microinjection induces changes in the transcriptome of bovine oocytes.

Gene knockdown techniques are widely used to examine the function of specific genes or proteins. While a variety of techniques are available, a technique commonly used on mammalian oocytes is mRNA knockdown by microinjection of small interfering RNA (siRNA), with non-specific siRNA injection used as a technical control. Here, we investigate whether and how the microinjection procedure itself affects the transcriptome of bovine oocytes. Injection of non-specific siRNA resulted in differential expression of 119 transcripts, of which 76 were down-regulated. Gene ontology analysis revealed that the differentially regulated genes were enriched in the biological processes of ATP synthesis, molecular transport and regulation of protein polyubiquitination. This study establishes a background effect of the microinjection procedure that should be borne in mind by those using microinjection to manipulate gene expression in oocytes.

Modelling human embryogenesis: embryo-like structures spark ethical and policy debate.

BACKGROUND: Studying the human peri-implantation period remains hindered by the limited accessibility of the in vivo environment and scarcity of research material. As such, continuing efforts have been directed towards developing embryo-like structures (ELS) from pluripotent stem cells (PSCs) that recapitulate aspects of embryogenesis in vitro. While the creation of such models offers immense potential for studying fundamental processes in both pre- and early post-implantation development, it also proves ethically contentious due to wide-ranging views on the moral and legal reverence due to human embryos. Lack of clarity on how to qualify and regulate research with ELS thus presents a challenge in that it may either limit this new field of research without valid grounds or allow it to develop without policies that reflect justified ethical concerns. OBJECTIVE AND RATIONALE: The aim of this article is to provide a comprehensive overview of the existing scientific approaches to generate ELS from mouse and human PSCs, as well as discuss future strategies towards innovation in the context of human development. Concurrently, we aim to set the agenda for the ethical and policy issues surrounding research on human ELS. SEARCH METHODS: The PubMed database was used to search peer-reviewed articles and reviews using the following terms: 'stem cells', 'pluripotency', 'implantation', 'preimplantation', 'post-implantation', 'blastocyst', 'embryoid bodies', 'synthetic embryos', 'embryo models', 'self-assembly', 'human embryo-like structures', 'artificial embryos' in combination with other keywords related to the subject area. The PubMed and Web of Science databases were also used to systematically search publications on the ethics of ELS and human embryo research by using the aforementioned keywords in combination with 'ethics', 'law', 'regulation' and equivalent terms. All relevant publications until December 2019 were critically evaluated and discussed. OUTCOMES: In vitro systems provide a promising way forward for uncovering early human development. Current platforms utilize PSCs in both two- and three-dimensional settings to mimic various early developmental stages, including epiblast, trophoblast and amniotic cavity formation, in addition to axis development and gastrulation. Nevertheless, much hinges on the term 'embryo-like'. Extension of traditional embryo frameworks to research with ELS reveals that (i) current embryo definitions require reconsideration, (ii) cellular convertibility challenges the attribution of moral standing on the basis of 'active potentiality' and (iii) meaningful application of embryo protective directives will require rethinking of the 14-day culture limit and moral weight attributed to (non-)viability. Many conceptual and normative (dis)similarities between ELS and embryos thus remain to be thoroughly elucidated. WIDER IMPLICATIONS: Modelling embryogenesis holds vast potential for both human developmental biology and understanding various etiologies associated with infertility. To date, ELS have been shown to recapitulate several aspects of peri-implantation development, but critically, cannot develop into a fetus. Yet, concurrent to scientific innovation, considering the extent to which the use of ELS may raise moral concerns typical of human embryo research remains paramount. This will be crucial for harnessing the potential of ELS as a valuable research tool, whilst remaining within a robust moral and legal framework of professionally acceptable practices.

Sperm DNA damage causes genomic instability in early embryonic development.

Genomic instability is common in human embryos, but the underlying causes are largely unknown. Here, we examined the consequences of sperm DNA damage on the embryonic genome by single-cell whole-genome sequencing of individual blastomeres from bovine embryos produced with sperm damaged by γ-radiation. Sperm DNA damage primarily leads to fragmentation of the paternal chromosomes followed by random distribution of the chromosomal fragments over the two sister cells in the first cell division. An unexpected secondary effect of sperm DNA damage is the induction of direct unequal cleavages, which include the poorly understood heterogoneic cell divisions. As a result, chaotic mosaicism is common in embryos derived from fertilizations with damaged sperm. The mosaic aneuploidies, uniparental disomies, and de novo structural variation induced by sperm DNA damage may compromise fertility and lead to rare congenital disorders when embryos escape developmental arrest.

PIWIL3 Forms a Complex with TDRKH in Mammalian Oocytes.

P-element induced wimpy testis (PIWIs) are crucial guardians of genome integrity, particularly in germ cells. While mammalian PIWIs have been primarily studied in mouse and rat, a homologue for the human PIWIL3 gene is absent in the Muridae family, and hence the unique function of PIWIL3 in germ cells cannot be effectively modeled by mouse knockouts. Herein, we investigated the expression, distribution, and interaction of PIWIL3 in bovine oocytes. We localized PIWIL3 to mitochondria, and demonstrated that PIWIL3 expression is stringently controlled both spatially and temporally before and after fertilization. Moreover, we identified PIWIL3 in a mitochondrial-recruited three-membered complex with Tudor and KH domain-containing protein (TDRKH) and poly(A)-specific ribonuclease-like domain containing 1 (PNLDC1), and demonstrated by mutagenesis that PIWIL3 N-terminal arginines are required for complex assembly. Finally, we sequenced the piRNAs bound to PIWIL3-TDRKH-PNLDC1 and report here that about 50% of these piRNAs map to transposable elements, recapitulating the important role of PIWIL3 in maintaining genome integrity in mammalian oocytes.

Initiation of X Chromosome Inactivation during Bovine Embryo Development.

X-chromosome inactivation (XCI) is a developmental process that aims to equalize the dosage of X-linked gene products between XY males and XX females in eutherian mammals. In female mouse embryos, paternal XCI is initiated at the 4-cell stage; however, the X chromosome is reactivated in the inner cell mass cells of blastocysts, and random XCI is subsequently initiated in epiblast cells. However, recent findings show that the patterns of XCI are not conserved among mammals. In this study, we used quantitative RT-PCR and RNA in situ hybridization combined with immunofluorescence to investigate the pattern of XCI during bovine embryo development. Expression of XIST (X-inactive specific transcript) RNA was significantly upregulated at the morula stage. For the first time, we demonstrate that XIST accumulation in bovine embryos starts in nuclei of female morulae, but its colocalization with histone H3 lysine 27 trimethylation was first detected in day 7 blastocysts. Both in the inner cell mass and in putative epiblast precursors, we observed a proportion of cells with XIST RNA and H3K27me3 colocalization. Surprisingly, the onset of XCI did not lead to a global downregulation of X-linked genes, even in day 9 blastocysts. Together, our findings confirm that diverse patterns of XCI initiation exist among developing mammalian embryos.

Cellular Fragments in the Perivitelline Space Are Not a Predictor of Expanded Blastocyst Quality.

The presence of cellular fragments in the perivitelline space is a commonly used parameter to determine quality before transfer of in vitro produced (IVP) embryos. However, this parameter is difficult to assess after blastocyst expansion. In this study, we used mechanical hatching to confirm the presence of cellular fragments in the perivitelline space of bovine IVP blastocysts. We further looked for associations between possible apoptosis within extruded cells/ cellular fragments and the quality of bovine blastocysts using quantitative RT-PCR and immunofluorescence. Surprisingly, more than 42% of expanded blastocysts had cellular fragments in the perivitelline space; however, more than 37% of extruded cells were TUNEL negative. We observed no significant difference in embryo quality between expanded blastocysts with and without cellular fragments in the perivitelline space. Overall, our data suggest that embryos extrude abnormal cells to maintain their developmental potential. The presence of fragmented cells is not an indicator of embryo quality.