Mammalian Preimplantation Embryos Research Articles

Common modes of ERK induction resolve into context-specific signalling via emergent networks and cell-type-specific transcriptional repression.

Fibroblast Growth Factor signalling via ERK exerts diverse roles in development and disease. In mammalian preimplantation embryos and naïve pluripotent stem cells ERK promotes differentiation, whereas in primed pluripotent states closer to somatic differentiation ERK sustains self-renewal. How can the same pathway produce different outcomes in two related cell types? To explore context-dependent ERK signalling we generated cell and mouse lines that allow for tissue- and time-specific ERK activation. Using these tools, we find that specificity in ERK response is mostly mediated by repression of transcriptional targets that occur in tandem with reductions in chromatin accessibility at regulatory regions. Furthermore, immediate early ERK responses are largely shared by different cell types but produce cell-specific programmes as these responses interface with emergent networks in the responding cells. Induction in naïve pluripotency is accompanied by chromatin changes, whereas in later stages it is not, suggesting that chromatin context does not shape signalling response. Altogether, our data suggest that cell-type-specific responses to ERK signalling exploit the same immediate early response, but then sculpt it to specific lineages via repression of distinct cellular programmes.

Evolution of Media Supporting the Development of Mammalian Preimplantation Embryos In Vitro.

Assisted reproductive technology has revolutionized our ability to genetically manipulate, maintain and rederive laboratory animals of biomedical importance; manipulate animal reproduction or genetics to boost production of farm animals; and improve human reproductive health. The media for in vitro manipulation and the culture of embryos play a critical role in the development of assisted reproductive technology. In this review, the evolution of culture media supporting embryo development in vitro from selected animal species, laboratory animals (mice and rats) and farm animals (pigs and cattle), will be discussed with a focus on the development of chemically defined media.

Transcriptome analysis of the effects of high temperature on zygotic genome activation in porcine embryos

Damage to the development of porcine gametes and embryos caused by high temperatures (HT) is one of the main reasons for the decline in the economic benefits of the livestock industry. Zygotic genome activation (ZGA) marks the beginning of gene expression programs in mammalian pre-implantation embryos. In pigs, ZGA occurs at the 4-cell (4 C) stage, indicating that correct gene expression at this stage plays an important regulatory role in embryonic development. However, the effect of the HT environment on early porcine embryonic development and the RNA expression profile of ZGA remain unclear. In this study, we compared the RNA transcription patterns of porcine 4 C embryos under normal and HT conditions using RNA-seq and identified 326 differentially expressed genes (DEGs). These changes were mainly related to DNA polymerase activity, DNA replication, and nucleotidyltransferase activity. In addition, entries for reverse transcription and endonuclease activity were enriched, indicating that ZGA interfered under HT conditions. Further comparison of the experimental results with the porcine ZGA gene revealed 39 ZGA genes among the DEGs. KEGG and GSEA analysis showed that the oxidative phosphorylation pathway was significantly enriched and signaling pathways related to energy metabolism were significantly downregulated. We also found that NDUFA6 and CDKN1A were located at the center of the protein-protein interaction network diagram of the DEGs. In summary, HT conditions affect mitochondrial function and oxidative phosphorylation levels, and lead to changes in the expression pattern of ZGA in early porcine embryos, with its hub genes NDUFA6 and CDKN1A.

P-112 Magnetic resonance spectroscopy at the micro scale (Micro-MRS) for non-invasively assessing individual mammalian embryos’ metabolism

Abstract Study question Can Micro-Magnetic Resonance Spectroscopy (micro-MRS) reveal the metabolic fingerprint of mammalian pre-implantation embryo? Summary answer Our research demonstrates that rapid, non-invasive analysis of embryos using Micro-Magnetic Resonance Spectroscopy (micro-MRS) can effectively uncover the significance of embryonic metabolites during development. What is known already Non-invasive selection of optimal embryos for transfer is a crucial challenge in assisted reproduction for both humans and animals. Magnetic Resonance Spectroscopy (MRS) emerges as a promising solution due to its chemical sensitivity and non-invasive nature. Despite its effectiveness in analysing large organisms, MRS faces obstacles in small sample handling and sensitivity, particularly with embryos, limiting its clinical and research applications. Our study overcomes these challenges using innovative microchip-based MRS sensors. This approach enabled successful MRS at the embryo scale, revealing metabolic biomarker linked to developmental potential in pre-implantation bovine embryos and opening new possibilities in assisted reproduction technology. Study design, size, duration Multiple MRS measuring sessions were conducted on two distinct embryo cohorts. The first cohort involved naturally arrested IVP embryos at early (8-cell) and late (morula/blastocyst) stages, establishing metabolic biomarkers in mammalian pre-implantation embryos. The second cohort involved 8-cell embryos which were further cultured over 10 days post-MRS with imaging at specific intervals (3 hours post-thawing, days 5, 8, and 10). This step was crucial to back-correlate biomarkers with in-vitro development based on bright field microscopy. Participants/materials, setting, methods Both studies involved vitrified samples that were subjected to a 50-minute MRS measurement following a 3-hour post-warming culture period. Single bovine embryo analysis was performed using our 1nL micro-MRS probe. Pre-implantation bovine embryos were obtained through IVF (n = 32 samples for cohort one and, n = 61 for cohort two). Post-MRS, the embryos were cultured in equilibrated IVC medium in a humidified incubator with 5% CO2 and 6% O2 at 38 °C. Statistical significance was set at p < 0.05. Main results and the role of chance Micro-Magnetic Resonance Spectroscopy (Micro-MRS) was utilized to identify metabolic markers in single bovine embryos, providing insights into their pre-implantation developmental stages. The spectroscopy data revealed distinct peak regions in the spectra of both cohorts, indicative of essential life-supporting fatty acids. Notably, up to six lipid markers were identified in the first cohort, five of which displayed statistically significant differences (p < 0.05). However, the majority of these biomarkers in the first cohort did not show significant expression differences in the groups of embryos assigned to the second cohort. An exception was noted in the case of the Principal Lipid Component (PLC) marker, which was significantly less prevalent in the arrested embryos than in those progressing to the blastocyst stage (p = 0.0016). Further data analysis based on AI-classification algorithms revealed up to 80% precision in predicting the development to the blastocyst stage solely based on their metabolic profiles at the 8-cell stage. Limitations, reasons for caution Magnetic Resonance Spectroscopy (MRS) shows promise for clinical and research use, requiring reliable signal detection from cellular components. While suitable for research, clinical deployment demands further safety assessments and protocol refinement. Additionally, larger sample sizes and data on post-MRS implantation and pregnancy rates are needed to validate AI-classification algorithms. Wider implications of the findings Micro-Magnetic Resonance Spectroscopy (Micro-MRS) holds potential as a reliable and non-invasive method for embryo screening prior to transfer. This innovative tool can shed light on the role of embryonic metabolites during development making Micro-MRS a key instrument in advancing both embryology research and human assisted reproduction techniques. Trial registration number not applicable

P-190 Impact of low lactate culture media in embryo development and IVF outcomes

Abstract Study question Can low lactate culture media improve embryo utilization rate in artificial reproductive technology (ART) cycles? Summary answer Embryos cultured in low lactate media showed a significant improved blastocyst utilization rate compared to embryos cultured in standard culture media. What is known already Culture media plays a key role in the embryonic development in the IVF lab. Currently, different culture medias are commercially available, yet no consensus on the most suitable media composition has been reached. Recently, minimizing metabolic stress for the embryo to enhance implantation and pregnancy rates has been suggested. Research on the metabolism of mammalian preimplantation embryos revealed that excess lactate in the culture medium, in addition to pyruvate and glucose, can burden metabolic efficiency, as embryos naturally produce lactate during energy production. Study design, size, duration The prospective, monocentric study was designed and conducted at the Kinderwunsch Institut Schenk GmbH (Dobl, Austria). A total of 60 women with an average age of 32.5 undergoing ICSI (intracytoplasmic sperm injection) treatment were included. All of them had at least 6 mature (MII) oocytes at time of insemination. Sibling oocytes were cultured in two different culture medias and outcomes were compared. Patients undergoing preimplantation genetic testing were excluded. Participants/materials, setting, methods Mature (MII) oocytes of each patient (total 621 MII oocytes) were inseminated by ICSI and then equally split into two culture medias: standard (313 oocytes) and low lactate (308 oocytes) covered with mineral-oil overlay and incubated in time-lapse under the same conditions. A total of 428 embryos were obtained. Embryos were evaluated using morphokinetic criteria. The fertilization rate, embryo utilization rate, pregnancy rate and time to Blastocyst were analyzed. Main results and the role of chance No differences were observed in the number of fertilized oocytes (n = 211 and 217 in the standard and low lactate media, respectively). The overall embryo utilization rate was significantly different (p < 0.008) resulting in 52% in standard media and 64.3% using low lactate media. Interestingly, age stratification (18-30; 31-35; 36-41) revealed overall higher embryo utilization rates when using low lactate media in all age groups. Blastocyst utilization rate was significantly different (p < 0.007) 43,1% and 58,05% in the standard and low lactate media, respectively. Regarding the time to blastocyst, it was observed that in the standard culture media 80% of the blastocysts reached blastocyst stage on D + 5 whereas in the low lactate media 92,07% reached blastocyst stage on D + 5, which was also significantly different (p < 0.001). No differences in pregnancy and live birth rates were observed. Limitations, reasons for caution The number of patients may be seen as a study limitation. Results should be confirmed with a bigger sample size. Wider implications of the findings Low lactate media could help improve IVF outcomes due to higher embryo utilization rate leading to better cumulative pregnancy rates. Excessive lactate concentration in culture media could negatively influence embryo metabolism. Trial registration number not applicable

Maternal factor Trim75 contributes to zygotic genome activation program in mouse early embryos.

Zygotic genome activation (ZGA) is an important event in the early embryo development, and human embryo developmental arrest has been highly correlated with ZGA failure in clinical studies. Although a few studies have linked maternal factors to mammalian ZGA, more studies are needed to fully elucidate the maternal factors that are involved in ZGA. In this study, we utilized published single-cell RNA sequencing data from a Dux-mediated mouse embryonic stem cell to induce a 2-cell-like transition state and selected potential drivers for the transition according to an RNA velocity analysis. An overlap of potential candidate markers of 2-cell-like-cells identified in this research with markers generated by various data sets suggests that Trim75 is a potential driver of minor ZGA and may recruit EP300 and establish H3K27ac in the gene body of minor ZGA genes, thereby contributing to mammalian preimplantation embryo development.

Single-cell sequencing shows mosaic aneuploidy in most human embryos.

Mammalian preimplantation embryos often contain chromosomal defects that arose in the first divisions after fertilization and affect a subpopulation of cells - an event known as mosaic aneuploidy. In this issue of the JCI, Chavli et al. report single-cell genomic sequencing data for rigorous evaluation of the incidence and degree of mosaic aneuploidy in healthy human in vitro fertilization (IVF) embryos. Remarkably, mosaic aneuploidy occurred in at least 80% of human blastocyst-stage embryos, with often less than 20% of cells showing defects. These findings confirm that mosaic aneuploidy is prevalent in human embryos, indicating that the process is a widespread event that rarely has clinical consequences. There are major implications for preimplantation genetic testing of aneuploidy (PGT-A), a test commonly used to screen and select IVF embryos for transfer. The application and benefit of this technology is controversial, and the findings provide more cause for caution on its use.

Insights into embryonic chromosomal instability: mechanisms of DNA elimination during mammalian preimplantation development.

Mammalian preimplantation embryos often contend with aneuploidy that arose either by the inheritance of meiotic errors from the gametes, or from mitotic mis-segregation events that occurred following fertilization. Regardless of the origin, mis-segregated chromosomes become encapsulated in micronuclei (MN) that are spatially isolated from the main nucleus. Much of our knowledge of MN formation comes from dividing somatic cells during tumorigenesis, but the error-prone cleavage-stage of early embryogenesis is fundamentally different. One unique aspect is that cellular fragmentation (CF), whereby small subcellular bodies pinch off embryonic blastomeres, is frequently observed. CF has been detected in both in vitro and in vivo-derived embryos and likely represents a response to chromosome mis-segregation since it only appears after MN formation. There are multiple fates for MN, including sequestration into CFs, but the molecular mechanism(s) by which this occurs remains unclear. Due to nuclear envelope rupture, the chromosomal material contained within MN and CFs becomes susceptible to double stranded-DNA breaks. Despite this damage, embryos may still progress to the blastocyst stage and exclude chromosome-containing CFs, as well as non-dividing aneuploid blastomeres, from participating in further development. Whether these are attempts to rectify MN formation or eliminate embryos with poor implantation potential is unknown and this review will discuss the potential implications of DNA removal by CF/blastomere exclusion. We will also extrapolate what is known about the intracellular pathways mediating MN formation and rupture in somatic cells to preimplantation embryogenesis and how nuclear budding and DNA release into the cytoplasm may impact overall development.

Low-input lipidomics reveals lipid metabolism remodelling during early mammalian embryo development.

Lipids are indispensable for energy storage, membrane structure and cell signalling. However, dynamic changes in various categories of endogenous lipids in mammalian early embryonic development have not been systematically characterized. Here we comprehensively investigated the dynamic lipid landscape during mouse and human early embryo development. Lipid signatures of different developmental stages are distinct, particularly for the phospholipid classes. We highlight that the high degree of phospholipid unsaturation is a conserved feature as embryos develop to the blastocyst stage. Moreover, we show that lipid desaturases such as SCD1 are required for in vitro blastocyst development and blastocyst implantation. One of the mechanisms is through the regulation of unsaturated fatty-acid-mediated fluidity of the plasma membrane and apical proteins and the establishment of apical-basal polarity during development of the eight-cell embryo to the blastocyst. Overall, our study provides an invaluable resource about the remodelling of the endogenous lipidome in mammalian preimplantation embryo development and mechanistic insights into the regulation of embryogenesis and implantation by lipid unsaturation.

Bovine embryo production in vitro: evolution of culture media and commercial perspectives.

In vitro produced embryos exhibit lower viability compared to their in vivo counterparts. Mammalian preimplantation embryos have the ability to reach the blastocyst stage in diverse culture media, showcasing considerable metabolic adaptability, which complicates the identification of optimal developmental conditions. Despite embryos successfully progressing to the blastocyst stage, adaptation to suboptimal culture environments may jeopardize blastocyst viability, cryotolerance, and implantation potential. Enhancing our capacity to support preimplantation embryonic development in vitro requires a deeper understanding of fundamental embryo physiology, including preferred metabolic substrates and pathways utilized by high-quality embryos. Armed with this knowledge, it becomes achievable to optimize culture conditions to support normal, in vivo-like embryo physiology, mitigate adaptive stress, and enhance viability. The objective of this review is to summarize the evolution of culture media for bovine embryos, highlighting significant milestones and remaining challenges.

Lactate regulates major zygotic genome activation by H3K18 lactylation in mammals.

Lactate is present at a high level in the microenvironment of mammalian preimplantation embryos in vivo and in vitro. However, its role in preimplantation development is unclear. Here, we report that lactate is highly enriched in the nuclei of early embryos when major zygotic genome activation (ZGA) occurs in humans and mice. The inhibition of its production and uptake results in developmental arrest at the 2-cell stage, major ZGA failure, and loss of lactate-derived H3K18lac, which could be rescued by the addition of Lac-CoA and recapitulated by overexpression of H3K18R mutation. By profiling the landscape of H3K18lac during mouse preimplantation development, we show that H3K18lac is enriched on the promoter regions of most major ZGA genes and correlates with their expressions. In humans, H3K18lac is also enriched in ZGA markers and temporally concomitant with their expressions. Taken together, we profile the landscapes of H3K18lac in mouse and human preimplantation embryos, and demonstrate the important role for H3K18lac in major ZGA, showing that a conserved metabolic mechanism underlies preimplantation development of mammalian embryos.

Time-lapse observation of mouse preimplantation embryos using a simple closed glass capillary method

Time-lapse observation is a popular method for analyzing mammalian preimplantation embryos, but it often requires expensive equipment and skilled techniques. We previously developed a simply and costly embryo-culture system in a sealed tube that does not require a CO2 incubator. In the present study, we developed a new time-lapse observation system using our previous culture method and a glass capillary. Zygotes were placed in a glass capillary and sunk in oil for observation under a stereomicroscope. Warming the capillary using a thermoplate enabled most of the zygotes to develop into blastocysts and produce healthy offspring. This time-lapse observation system captured images every 30 min for up to 5 days, which confirmed that the developmental speed and quality of the embryos were not affected, even with fluorescence. Overall, this new system is a simple time-lapse observation method for preimplantation embryos that does not require dedicated machines and advanced techniques.

N4-acetylcytidine of Nop2 mRNA is required for the transition of morula-to-blastocyst.

N-acetyltransferase 10 (NAT10)-mediated N4-acetylcytidine (ac4C) modification is crucial for mRNA stability and translation efficiency, yet the underlying function in mammalian preimplantation embryos remains unclear. Here, we characterized the ac4C modification landscape in mouse early embryos and found that the majority of embryos deficient in ac4C writer-NAT10 failed to develop into normal blastocysts. Through single-cell sequencing, RNA-seq, acetylated RNA immunoprecipitation combined with PCR (acRIP-PCR), and embryonic phenotype monitoring, Nop2 was screened as a target gene of Nat10. Mechanistically, Nat10 knockdown decreases the ac4C modification on Nop2 mRNA and reduces RNA and protein abundance by affecting the mRNA stability of Nop2. Then, depletion of NOP2 may inhibit the translation of transcription factor TEAD4, resulting in defective expression of the downstream lineage-specific gene Cdx2, and ultimately preventing blastomeres from undergoing the trophectoderm (TE) fate. However, exogenous Nop2 mRNA partially reverses this abnormal development. In conclusion, our findings demonstrate that defective ac4C modification of Nop2 mRNA hinders the morula-to-blastocyst transition by influencing the first cell fate decision in mice.

NANOG expression in parthenogenetic porcine blastocysts is required for intact lineage specification and pluripotency.

Nanog homeobox (NANOG) is a core transcription factor that contributes to pluripotency along with octamer binding transcription factor-4 (OCT4) and sex determining region-Y box-2 (SOX2). It is an epiblast lineage marker in mammalian pre-implantation embryos and exhibits a species-specific expression pattern. Therefore, it is important to understand the lineage of NANOG, the trophectoderm, and the primitive endoderm in the pig embryo. A loss- and gain-of-function analysis was done to determine the role of NANOG in lineage specification in parthenogenetic porcine blastocysts. We analyzed the relationship between NANOG and pluripotent core transcription factors and other lineage makers. In NANOG-null late blastocysts, OCT4-, SOX2-, and SOX17-positive cells were decreased, whereas GATA binding protein 6 (GATA6)-positive cells were increased. Quantitative real-time polymerase chain reaction revealed that the expression of SOX2 was decreased in NANOG-null blastocysts, whereas that of primitive endoderm makers, except SOX17, was increased. In NANOG-overexpressing blastocysts, caudal type homeobox 2 (CDX2-), SOX17-, and GATA6-positive cells were decreased. The results indicated that the expression of primitive endoderm markers and trophectoderm-related genes was decreased. Taken together, the results demonstrate that NANOG is involved in the epiblast and primitive endoderm differentiation and is essential for maintaining pluripotency within the epiblast.

The mammalian preimplantation embryo: Its role in the environmental programming of postnatal health and performance

During formation of the preimplantation embryo several cellular and molecular milestones take place, making the few cells forming the early embryo vulnerable to environmental stressors than can impair epigenetic reprogramming and controls of gene expression. Although these molecular alterations can result in embryonic death, a significant developmental plasticity is present in the preimplantation embryo that promotes full-term pregnancy. Prenatal epigenetic modifications are inherited during mitosis and can perpetuate specific phenotypes during early postnatal development and adulthood. As such, the preimplantation phase is a developmental window where developmental programming can take place in response to the embryonic microenvironment present in vivo or in vitro. In this review, the relevance of the preimplantation embryo as a developmental stage where offspring health and performance can be programmed is discussed, with emphasis on malnutrition and assisted reproductive technologies; two major environmental insults with important implications for livestock production and human reproductive medicine.

Dynamic nucleosome remodeling mediated by YY1 underlies early mouse development.

Nucleosome positioning can alter the accessibility of DNA-binding proteins to their cognate DNA elements, and thus its precise control is essential for cell identity and function. Mammalian preimplantation embryos undergo temporal changes in gene expression and cell potency, suggesting the involvement of dynamic epigenetic control during this developmental phase. However, the dynamics of nucleosome organization during early development are poorly understood. In this study, using a low-input MNase-seq method, we show that nucleosome positioning is globally obscure in zygotes but becomes well defined during subsequent development. Down-regulation of the chromatin assembly in embryonic stem cells can partially reverse nucleosome organization into a zygote-like pattern, suggesting a possible link between the chromatin assembly pathway and fuzzy nucleosomes in zygotes. We also reveal that YY1, a zinc finger-containing transcription factor expressed upon zygotic genome activation, regulates the de novo formation of well-positioned nucleosome arrays at the regulatory elements through identifying YY1-binding sites in eight-cell embryos. The YY1-binding regions acquire H3K27ac enrichment around the eight-cell and morula stages, and YY1 depletion impairs the morula-to-blastocyst transition. Thus, our study delineates the remodeling of nucleosome organization and its underlying mechanism during early mouse development.

CircKDM5B sponges miR-128 to regulate porcine blastocyst development by modulating trophectoderm barrier function.

Circular RNAs (circRNAs), which exert critical functions in the regulation of transcriptional and posttranscriptional gene expression, are found in mammalian cells but their functions in mammalian preimplantation embryo development remain poorly understood. Here, we showed that circKDM5B mediated miRNA128 to regulate porcine early embryo development. We screened circRNAs potentially expressed in porcine embryos by integrated analysis of sequencing data from mouse and human embryos as well as porcine oocytes. An authentic circRNA originating from histone demethylase KDM5B (referred to as circKDM5B) was abundantly expressed in porcine embryos. Functional studies revealed that circKDM5B knockdown not only significantly reduced blastocyst formation but also decreased the number of total cells and trophectoderm (TE) cells. Moreover, the knockdown of circKDM5B resulted in the disturbance of tight junction assembly and impaired paracellular sealing within the TE epithelium. Mechanistically, miR-128 inhibitor injection could rescue the early development of circKDM5B knockdown embryos. Taken together, the findings revealed that circKDM5B functions as a miR-128 sponge, thereby facilitating early embryonic development in pigs through the modulation of gene expression linked to tight junction assembly.

Multi-level Fgf4- and apoptosis-dependent regulatory mechanism ensures the plasticity of ESC-chimaeric mouse embryo.

The preimplantation mammalian (including mouse and human) embryo holds remarkable regulatory abilities, which have found their application for example in the preimplantation genetic diagnosis of human embryos. Another manifestation of this developmental plasticity is the possibility of obtaining chimaeras by combining either two embryos or embryos and pluripotent stem cells, which enables the verification of the cell pluripotency and generation of genetically modified animals used to elucidate gene function. Using mouse chimaeric embryos (constructed by injection of embryonic stem cells into the 8-cell embryos) as a tool, we aimed to explore the mechanisms underlying the regulatory nature of the preimplantation mouse embryo. We comprehensively demonstrated the functioning of a multi-level regulatory mechanism involving the FGF4/MAPK signalling as a leading player in the communication between both components of the chimaera. This pathway, coupled with the apoptosis, cleavage division pattern, and cell cycle duration controlling the size of ESC component and giving it a competitive advantage over host embryo blastomeres, provides a cellular and molecular basis for regulative development, ensuring the generation of the embryo characterised by proper cellular composition.

Absence of a molecular circadian clock in the preimplantation embryo is a conserved characteristic in the mammal.

It is not known when a functional circadian clock is established in the developing embryo. Lack of expression of key genes involved in the clock mechanism is indicative that a functional circadian clock mechanism is absent in the mammalian preimplantation embryo through the blastocyst stage of development. An embryonic circadian clock could conceivably organize cellular and developmental events temporally and in synchrony with other circadian rhythms in the mother. The hypothesis that a functional molecular clock exists in the preimplantation bovine, pig, human, and mouse embryo was tested by using publicly available RNAseq datasets to examine developmental changes in expression of the core genes responsible for the circadian clock - CLOCK, ARNTL, PER1, PER2, CRY1, and CRY2. In general, the transcript abundance of each gene decreased as development advanced to the blastocyst stage. The most notable exception was for CRY2, where transcript abundance was low and constant from the two-cell or four-cell to the blastocyst stage. Developmental patterns were generally the same for all species although there were some species-specific patterns such as an absence of PER1 expression in the pig, an increase in ARNTL expression at the four-cell stage in human, and an increase in expression of Clock and Per1 from the zygote to two-cell stage in the mouse. Analysis of intronic reads (indicative of embryonic transcription) for bovine embryos indicated an absence of embryonic transcription. Immunoreactive CRY1 was not detected in the bovine blastocyst. Results indicate that the preimplantation mammalian embryo lacks a functional intrinsic clock although specific components of the clock mechanism could conceivably play a role in other functions in the embryo.

P-127 Micro-Magnetic Resonance Spectroscopy of individual mammalian embryos: a safe and non-invasive diagnostic tool for embryo screening in assisted reproduction

Abstract Study question Is micro-magnetic resonance (micro-MRS) a safe tool to non-invasively unravel the metabolic fingerprint of single mammalian embryos? Summary answer We successfully tested the safety of micro-MRS in a mouse model. No long or short-term adverse effect was found in vitro and in vivo. What is known already Non-invasive selection of the best embryo to transfer is one of the most significant challenges for ART professionals. The chemical sensitivity, resolving power, and, more importantly, the non-invasive nature of MRS makes it an excellent candidate to investigate the building blocks of complex organisms. Although MRS is a well-established technique for the biochemical profiling of large organisms, handling small samples like embryos and 3D cell cultures and spheroids alongside sensitivity issues has prevented its adoption for clinical and research applications. Our group has overcome these limitations with microchip-based sensors to leverage non-invasive MRS technology down to the embryo scale. Study design, size, duration This safety study was divided into two main phases in order to test all the components needed to operate the micro-MRS analysis. In phase 1 we used > 800 2-cell embryos to test a) radiofrequency exposure, b) device activation, and c) static magnetic field exposure. In phase 2 we confirmed in-vivo that MF exposure was not affecting live animals (n267) over 3 generations of mice by assessing different IVF outcomes, natural mating, live parameters and histopathology. Participants/materials, setting, methods Cryopreserved 2-cell B6D2F1 mouse embryos were purchased cryopreserved from JanvierLabs. In phase1 embryos were thawed and used for testing micro-MRS main components' safety. Mouse embryo assay (MEA) was used to first assess micro-MRS adverse effects in-vitro. In phase 2 embryos were exposed to magnetic field in a 9.4T Magnet for 1h at 37 °C, then surgically transferred to surrogate mothers. Both mothers and progeny were tracked up to F3. Statistical significance was set at p < 0.05. Main results and the role of chance This is the first study ever conducted to assess the safety and efficacy of micro-MRS on pre-implantation mammalian embryos. Our results show, with statistical significance, that micro-MRS is safe for use in mammalians. In particular, we have de-risked the three main aspects/components that could affect the embryos: 1) Materials to which the embryos are exposed when loaded into the device, 2) Radiofrequency energy required to activate the device and perform a measurement, and 3) Magnetic field required to analyze the embryos biochemistry in a non-invasive way. Overall no adverse effect was observed when the primary IVF outcomes were assessed, such as implantation rate and live birth rate. In addition, no visible alteration of the overall appearance of ED14.5 foetuses, F1, F2 and F3 mice was observed when comparing the mice obtained from treated or control embryos. The surrogate mothers did not display any significant alteration or visible health issues after embryo transfer. F1 and F2 mice were able to naturally mate and conceive new progeny suggesting that the reproductive potential of the animals is not affected by the magnetic field exposure. Furthermore, histopathological analysis of 5 representative organs did not reveal adverse effects on both surrogate mothers or F1 pups. Limitations, reasons for caution A vital step towards establishing MRS as a clinical and research tool is the reliable detection of a wide range of signals from cellular components. Our method is ready for R&D studies, while its clinical application requires further safety studies and protocol optimization. Wider implications of the findings A non-invasive quick assay would provide the means to reveal the role of embryonic lipids throughout development. Micro-MRS can further develop into a safe embryo assay for selection before embryo transfer. This would apply to both human and animal ART, whose success rate is relatively low. Trial registration number NA