Failures during conceptus elongation are a major cause of pregnancy losses in ungulates, exerting a relevant economic impact on farming. The developmental events occurring during this period are poorly understood, mainly because this process cannot be recapitulated in vitro. Previous studies have established an in vitro post-hatching development (PHD) system that supports bovine embryo development beyond the blastocyst stage, based on agarose gel tunnels and serum and glucose-enriched medium. Unfortunately, under this system embryonic disc formation is not achieved and embryos show notorious signs of apoptosis and necrosis. The objective of this study has been to develop an in vitro system able to support embryonic disc formation. We first compared post-hatching development inside agarose tunnels or free-floating over an agarose-coated dish in serum and glucose-enriched medium (PHD medium). Culture inside agarose tunnels shaped embryo morphology by physical constriction, but it restricted embryo growth and did not provide any significant advantage in terms of development of hypoblast and epiblast lineages. In contrast to PHD medium, a chemically defined and enriched medium (N2B27) supported complete hypoblast migration and epiblast survival in vitro, even in the absence of agarose coating. Cells expressing the pluripotency marker SOX2 were observed in ~56 % of the embryos and ~25 % developed embryonic disc-like structures formed by SOX2+ cells. In summary, here we provide a culture system that supports trophectoderm proliferation, hypoblast migration and epiblast survival after the blastocyst stage.

Targeted knock-in (KI) can be achieved in embryos by clustered regularly interspaced short palindromic repeats (CRISPR)-assisted homology directed repair (HDR). However, HDR efficiency is constrained by the competition of nonhomologous end joining.The objective of this study was to explore whether CRISPR-assisted targeted KI rates can be improved in bovine embryos by exposure to the HDR enhancer RS-1. In vitro produced zygotes were injected with CRISPR components (300 ng/µl Cas9 messenger RNA and 100 ng/µl single guide RNA against a noncoding region) and a singlestranded DNA (ssDNA) repair template (100 ng/µl). ssDNA template contained a 6 bp XbaI site insert, allowing targeted KI detection by restriction analysis, flanked by 50 bp homology arms. Following microinjection, zygotes were exposed to 0, 3.75, or 7.5 µM RS-1 for 24 hr. No differences were noted between groups in terms of development or genome edition rates. However, targeted KI rates were doubled in the group exposed to 7.5 µM RS-1 compared to the others (52.8% vs. 25% and 23.1%, for 7.5, 0, and 3.75 µM, respectively). In conclusion, transient exposure to 7.5 µM RS-1 enhances targeted KI rates resulting in approximately half of the embryos containing the intended mutation, hence allowing direct KI generation in embryos.

STUDY QUESTION Is relative mitochondrial DNA (mtDNA) content in cumulus cells related to embryo developmental competence in humans and/or the bovine model? SUMMARY ANSWER mtDNA content in cumulus cells provides poor predictive value of oocyte developmental potential, both in vitro and following embryo transfer. WHAT IS KNOWN ALREADY Cumulus cells are closely connected to the oocyte through transzonal projections, serving essential metabolic functions during folliculogenesis. These oocyte-supporting cells are removed and discarded prior to ICSI, thereby providing interesting biological material on which to perform molecular analyses designed to identify markers that predict oocyte developmental competence. Previous studies have positively associated oocyte mtDNA content with developmental potential in animal models and women. However, it remains debatable whether mtDNA content in cumulus cells could be used as a proxy to infer oocyte developmental potential STUDY DESIGN, SIZE, DURATION mtDNA content was analyzed in cumulus cells obtained from 109 human oocytes unable to develop to blastocyst, able to develop to blastocyst but failing to establish pregnancy, or able to develop to blastocyst and to establish pregnancy. mtDNA analysis was also performed on bovine cumulus samples collected from 120 oocytes unable to cleave, oocytes developing into cleaved embryos but arresting development prior to the blastocyst stage, or oocytes developing to blastocysts. PARTICIPANTS/MATERIALS, SETTING, METHODS Human cumulus cells samples were obtained from women undergoing IVF. Only unfrozen oocytes and embryos not submitted to preimplantation genetic testing were included in the analysis. Bovine samples were obtained from slaughtered cattle and individually matured, fertilized and cultured in vitro. Relative mtDNA was assessed by quantitative PCR analysis. MAIN RESULTS AND THE ROLE OF CHANCE mtDNA content in human and bovine cumulus cells did not differ according to the developmental potential of their enclosed oocyte. Moreover, mtDNA content in bovine oocytes did not correlate with that of their corresponding cumulus cells. LARGE SCALE DATA N/A LIMITATIONS, REASONS FOR CAUTION The lack of correlation found between mtDNA content in human cumulus cells and oocytes was also assessed in bovine samples. Although bovine folliculogenesis, monoovulatory ovulation and early embryo development exhibit considerable similarities with that of humans, they may not be fully comparable. WIDER IMPLICATIONS OF THE FINDINGS The use of molecular markers for oocyte developmental potential in cumulus cells could be used to enhance success rates following single embryo transfer. However, our data indicate that mtDNA in cumulus cells is not a good proxy for oocyte quality. STUDY FUNDING/COMPETING INTEREST(S) Research was supported by the Industrial Doctorate Project IND2017/BIO-7748 funded by Madrid Region Government. The authors declare no competing interests.

Cumulus cells provide an interesting biological material to perform analyses to understand the molecular clues determining oocyte competence. The objective of this study was to analyze the transcriptional differences between cumulus cells from oocytes exhibiting different developmental potentials following individual in vitro embryo production by RNA‐seq. Cumulus cells were allocated into three groups according to the developmental potential of the oocyte following fertilization: (1) oocytes developing to blastocysts (Bl+), (2) oocytes cleaving but arresting development before the blastocyst stage (Bl−), and (3) oocytes not cleaving (Cl−). RNAseq was performed on 4 (Cl−) or 5 samples (Bl+ and Bl−) of cumulus cells pooled from 10 cumulus‐oocyte complexes per group. A total of 49, 50, and 18 differentially expressed genes (DEGs) were detected in the comparisons Bl+ versus Bl−, Bl+ versus Cl− and Bl‐ versus Cl−, respectively, showing a fold change greater than 1.5 at an adjusted p value <0.05. Focussing on DEGs in cumulus cells from Bl+ group, 10 DEGs were common to both comparisons (10/49 from Bl+ vs. Bl−, 10/50 from Bl+ vs. Cl−). These DEGs correspond to 6 upregulated genes (HBE1, ITGA1, PAPPA, AKAP12, ITGA5, and SLC1A4), and 4 downregulated genes (GSTA1, PSMB8, FMOD, and SFRP4) in Bl+ compared to the other groups, from which 7 were validated by quantitative PCR (HBE1, ITGA1, PAPPA, AKAP12, ITGA5, PSMB8 and SFRP4). These genes are involved in critical biological functions such as integrin‐mediated cell adhesion, oxygen availability, IGF and Wnt signaling or PKA pathway, highlighting specific biological processes altered in incompetent in vitro maturation oocytes.