PFOS and F-53B disrupted inner cell mass development in mouse preimplantation embryo.

Perfluorooctane sulfonic acid (PFOS) is a perfluoroalkyl and polyfluoroalkyl substance (PFAS) widely used in daily life. As its toxicity was confirmed, it has been gradually substituted by F-53B (chlorinated polyfluoroalkyl sulfonates, Cl-PFESAs) in China. PFOS exposure during prenatal development may hinder the development of preimplantation embryos, as indicated by recent epidemiological research and in vivo assays. However, the embryotoxicity data for F-53B are scarce. Furthermore, knowledge about the toxicity of F-53B and PFOS exposure to internal follicular fluid concentrations on early preimplantation embryo development remains limited. In this study, internal exposure concentrations of PFOS (10 nM) and F-53B (2 nM) in human follicular fluid were chosen to study the effects of PFAS on early mouse preimplantation embryo development. We found that both PFOS and F-53B treated zygotes exhibited higher ROS activity in 8-cell embryos but not in 2-cell stage embryos. PFOS and F-53B significantly affected the proportion and aggregation of the inner cell mass (ICM) in the blastocyst, but not the total cell number. Mouse embryonic stem cells (mESCs, isolated from the ICM) and embryoid body (EB) assays were employed to assess the toxicity of PFOS and F-53B on the development and differentiation of embryonic pluripotent cells. These results suggested that mESCs exhibited more DNA damage and abnormal germ layer differentiation after brief exposure to PFOS or F-53B. Finally, RNA-sequencing revealed that PFOS and F-53B exposure affected mESCs biosynthetic processes and chromatin-nucleosome assembly. Our results indicate that F-53B has potential risks as an alternative to PFOS, which disrupts ICM development and differentiation.

Does Trophectoderm Mitochondrial DNA Content Affect Embryo Developmental and Implantation Potential?

A retrospective case control study was undertaken at the molecular biology department of a private center for reproductive medicine in order to determine whether any correlation exists between the mitochondrial DNA (mtDNA) content of trophectoderm and embryo developmental potential. A total of 275 couples underwent IVF treatment, producing a total of 716 embryos. The trophectoderm was biopsied from each embryo at the blastocyst stage (day 5 or day 6 post-fertilization) subjected to low-pass next-generation sequencing (NGS), for the purpose of detecting aneuploidy. For each sample, the number of mtDNA reads obtained after analysis using NGS was divided by the number of reads attributable to the nuclear genome. The mtDNA copy number was found to be higher in aneuploid embryos than in those that were euploid (mean mtDNA ratio ± SD: 1.13 ± 1.37 versus 1.45 ± 1.78, p = 0.02) and in day 5 biopsies compared to day 6 biopsies (1.41 ± 1.66 vs. 1.19 ± 1.27, p = 0.001), whereas no statistically significant differences in mtDNA content were seen in relation to embryo morphology (1.58 ± 2.44 vs. 2.19 ± 2.89, p = 0.12), genetic sex (1.27 ± 1.29 vs. 1.27 ± 1.18, p = 0.99), maternal age (1.31 ± 1.41 vs. 1.33 ± 1.29, p = 0.43), or its ability to implant (1.14 ± 0.88 vs. 1.21 ± 1.16, p = 0.39). mtDNA has small potential to serve as an additional, independent biomarker for embryo selection.

Evidence of haptoglobin in the porcine female genital tract during oestrous cycle and its effect on in vitro embryo production.

Recent evidence supports involvement of the acute phase protein haptoglobin in numerous events during mammalian reproduction. The present study represents an in-depth investigation of haptoglobin expression and secretion in the porcine oviduct and uterus, and assesses its effect on porcine in vitro embryo production. A systematic study was made of sows in different oestrous stages: late follicular, early luteal and late luteal stages. Relative haptoglobin mRNA abundance was quantified by RT-qPCR. In addition, expression of the protein was analysed by immunohistochemistry and the results were complemented by Western-blot and proteomic analyses of the oviductal and uterine fluids. In vitro porcine fertilization and embryo culture were carried out in the presence of haptoglobin. The results indicate that haptoglobin mRNA expression in the porcine oviduct and uterus is most abundant during the late luteal stage of the oestrous cycle. By means of Western blot and proteomic analyses haptoglobin presence was demonstrated in the oviduct epithelium and in the oviductal and uterine fluids in different stages of the oestrous cycle. The addition of haptoglobin during gamete co-incubation had no effect on sperm penetration, monospermy or efficiency rates; however, compared with the control group, blastocyst development was significantly improved when haptoglobin was present (haptoglobin: 64.50% vs. control: 37.83%; p < 0.05). In conclusion, the presence of haptoglobin in the oviduct and uterus of sows at different stages of the oestrous cycle suggests that it plays an important role in the reproduction process. The addition of haptoglobin during in vitro embryo production improved the blastocyst rates.

Lipid profile of bovine grade-1 blastocysts produced either in vivo or in vitro before and after slow freezing process.

Currently, in vitro embryo production (IVP) is successfully commercially applied in cattle. However, the high sensitivity of embryos to cryopreservation in comparison to in vivo (IVD) embryos slows the dissemination of this biotechnology. Reduced cryotolerance is frequently associated with lipid accumulation in the cytoplasm mainly due to in vitro culture conditions. The objective of this study was to evaluate the lipid composition of biopsied and sexed embryos, produced either in vivo or in vitro from the same Holstein heifers before and after a slow freezing protocol. Lipid extracts were analysed by liquid chromatography-high resolution mass spectrometry, which enabled the detection of 496 features. Our results highlighted a lipid enrichment of IVP embryos in triglycerides and oxidised glycerophospholipids and a reduced abundance in glycerophospholipids. The slow freezing process affected the lipid profiles of IVP and IVD embryos similarly. Lysophosphatidylcholine content was reduced when embryos were frozen/thawed. In conclusion, the embryonic lipid profile is impacted by IVP and slow freezing protocols but not by sex. Lysophosphatidylcholine seemed highly sensitive to cryopreservation and might contribute to explain the lower quality of frozen embryos. Further studies are required to improve embryo freezability by modulating the lipidome.

Eicosapentaenoic acid supplemented to in vitro maturation medium results in lesser lipid content and intracellular reactive oxygen species in blastocysts of cattle.

Sub-optimal cattle embryo development to the blastocyst stage still is a problem when conducting in vitro production (IVP) procedures. Supplementation of in vitro maturation (IVM) medium with omega 3-polyunsaturated eicosapentaenoic acid (EPA) is an approach that might have positive effects on lipid metabolism of cattle oocytes, potentially improving subsequent embryo development. The aim of this study was to evaluate effects of EPA addition to serum-free IVM medium on pronuclear formation after in vitro fertilization, cleavage, and blastocyst rates. Effects of EPA on lipid accumulation and intracellular reactive oxygen species (ROS) generation with IVP of cattle embryos was also investigated. In all experiments, cumulus-oocyte complexes were matured in IVM medium supplemented with 0 nM, 1 nM, or 1 µM EPA for 24 h. Pronuclear formation, cleavage, and blastocyst rates were similar for embryos when there was supplementation of EPA at all concentrations to those of the control group (P > 0.05). The inclusion of 1 nM EPA in medium resulted in a greater lipid content and less intracellular ROS in day 8-embryos compared with those of the Control group (P < 0.05). There were no differences, however, when there was inclusion of 1 µM EPA compared to embryos of the Control group at the day 8 developmental stage (P > 0.05). In conclusion, supplementation with IVM medium with the 1 nM EPA concentration resulted in a lesser blastocyst lipid and intracellular ROS concentration, without modifying embryo development, therefore, EPA could be a desirable supplement to improve embryo quality in cattle.

ELOVL5 Participates in Embryonic Lipid Determination of Cellular Membranes and Cytoplasmic Droplets.

Embryonic lipids are crucial for the formation of cellular membranes and dynamically participate in metabolic pathways. Cells can synthesize simple fatty acids, and the elongation of fatty acids facilitates the formation of complex lipids. The aim of this work was to investigate the involvement of the elongation of very long chain fatty acid enzyme 5 (ELOVL5) in embryonic development and lipid determination. Bovine embryos were produced in vitro using a standard protocol and randomly divided to receive one of three treatments at Day 4: morpholino (Mo) gene expression knockdown assay for ELOVL5 (ELOVL5-Mo), Mo antisense oligonucleotides for the thalassemic ß-globulin human mRNA (technical control Mo), and placebo (biological control). The phenotypes of embryonic development, cell number, ELOVL5 protein abundance, lipid droplet deposits, and lipid fingerprint were investigated. No detrimental effects (p > 0.05) were observed on embryo development in terms of cleavage (59.4 ± 3.5%, 63.6 ± 4.1%, and 65.4 ± 2.2%), blastocyst production (31.3 ± 4.2%, 28.1 ± 4.9%, and 36.1 ± 2.1%), and blastocyst cell number (99.6 ± 7.7, 100.2 ± 6.2, 86.8 ± 5.6), respectively, for biological control, technical control Mo, and ELOVL5-Mo. ELOVL5 protein abundance and cytoplasmic lipid droplet deposition were increased (p < 0.05) in ELOVL5-Mo-derived blastocysts compared with the controls. However, seven lipid species, including phosphatidylcholines, phosphatidylethanolamines, and triacylglycerol, were downregulated in the ELOVL5-Mo-derived blastocysts compared with the biological control. Therefore, ELOVL5 is involved in the determination of embryonic lipid content and composition. Transient translational blockage of ELOVL5 reduced the expression of specific lipid species and promoted increased cytoplasmic lipid droplet deposition, but with no apparent deleterious effect on embryonic development and blastocyst cell number.

Metabolomic differences in blastocoel and uterine fluids collected in vivo by ultrasound biomicroscopy on rabbit embryos†.

The success of embryo development and implantation depends in part on the environment in which the embryo evolves. However, the composition of the uterine fluid surrounding the embryo in the peri-implantation period remains poorly studied. In this work, we aimed to develop a new strategy to visualize, collect, and analyze both blastocoelic liquid and juxta-embryonic uterine fluid from in vivo peri-implantation rabbit embryos. Using high-resolution ultrasound biomicroscopy, embryos were observed as fluid-filled anechoic vesicles, some of which were surrounded by a thin layer of uterine fluid. Ultrasound-guided puncture and aspiration of both the blastocoelic fluid contained in the embryo and the uterine fluid in the vicinity of the embryo were performed. Using nuclear magnetic resonance spectroscopy, altogether 24 metabolites were identified and quantified, of which 21 were detected in both fluids with a higher concentration in the uterus compared to the blastocoel. In contrast, pyruvate was detected at a higher concentration in blastocoelic compared to uterine fluid. Two acidic amino acids, glutamate and aspartate, were not detected in uterine fluid in contrast to blastocoelic fluid, suggesting a local regulation of uterine fluid composition. To our knowledge, this is the first report of simultaneous analysis of blastocoelic and uterine fluids collected in vivo at the time of implantation in mammals, shedding new insight for understanding the relationship between the embryo and its local environment at this critical period of development.

Analysis of accessible chromatin landscape in the inner cell mass and trophectoderm of human blastocysts.

Early embryonic development is characterized by drastic changes in chromatin structure that affects the accessibility of the chromatin. In human, the chromosome reorganization and its involvement in the first linage segregation are poorly characterized due to the difficulties in obtaining human embryonic material and limitation on low input technologies. In this study, we aimed to explore the chromatin remodeling pattern in human preimplantation embryos and gain insight into the epigenetic regulation of inner cell mass (ICM) and trophectoderm (TE) differentiation. We optimized ATAC-seq (an assay for transposase-accessible chromatin using sequencing) to analyze the chromatin accessibility landscape for low DNA input. Sixteen preimplantation human blastocysts frozen on Day 6 were used. Our data showed that ATAC peak distributions of the promoter regions (<1 kb) and distal regions versus other regions were significantly different between ICM versus TE samples (P < 0.01). We detected that a higher percentage of accessible binding loci were located within 1 kb of the transcription start site in ICM compared to TE (P < 0.01). However, a higher percentage of accessible regions was detected in the distal region of TE compared to ICM (P < 0.01). In addition, eight differential peaks with a false discovery rate <0.05 between ICM and TE were detected. This is the first study to compare the landscape of the accessible chromatin between ICM and TE of human preimplantation embryos, which unveiled chromatin-level epigenetic regulation of cell lineage specification in early embryo development.

Editing DNA Methylation in Mammalian Embryos.

DNA methylation in mammals is essential for numerous biological functions, such as ensuring chromosomal stability, genomic imprinting, and X-chromosome inactivation through transcriptional regulation. Gene knockout of DNA methyltransferases and demethylation enzymes has made significant contributions to analyzing the functions of DNA methylation in development. By applying epigenome editing, it is now possible to manipulate DNA methylation in specific genomic regions and to understand the functions of these modifications. In this review, we first describe recent DNA methylation editing technology. We then focused on changes in DNA methylation status during mammalian gametogenesis and preimplantation development, and have discussed the implications of applying this technology to early embryos.

Ambient Lipidomic Analysis of Single Mammalian Oocytes and Preimplantation Embryos Using Desorption Electrospray Ionization (DESI) Mass Spectrometry.

Desorption electrospray ionization (DESI) is a spray-based ambient ionization method for mass spectrometry (MS) that generates ions in native atmospheric conditions (e.g., pressure and temperature). Ambient ionization allows in situ analysis of unmodified samples by eliminating analyte extraction and separation steps before MS. Lipid analysis of individual mammalian oocytes and preimplantation embryos is challenging because of their complex chemical composition and minute dimensions (100-300 µm in diameter). Nonetheless, DESI-MS can provide comprehensive lipidomic profiles of individual oocytes or embryos using a fast and simple workflow. DESI-MS lipid profiles include many classes of lipids such as phosphatidylcholines (PC), triacylglycerols (TAG), free fatty acids (FFA), phosphatidylethanolamines (PE), phosphatidylinositols (PI), phosphatidylserines (PS), diacylglycerols (DAG), ubiquinone, cholesterol and cholesterol derivatives (e.g., cholesterol sulfate and cholesterol esters). Depending on the mass spectrometer used, there is the additional possibility of obtaining structural information of lipids via MSn, and molecular formulae via high resolution MS. Here, we describe the procedures for sample handling, the DESI-MS experimental arrangement, and the data collection and data analysis using low and high-resolution mass spectrometers (such as a linear ion trap and Orbitrap mass analyzer, respectively), as well as strategies for structural characterization of lipids. Lastly, we detail our workflow for relating the chemical information obtained by DESI-MS into the biological context of oocyte and embryo metabolism.

Polycomb Group Proteins Regulate Chromatin Architecture in Mouse Oocytes and Early Embryos.

In mammals, chromatin organization undergoes drastic reorganization during oocyte development. However, the dynamics of three-dimensional chromatin structure in this process is poorly characterized. Using low-input Hi-C (genome-wide chromatin conformation capture), we found that a unique chromatin organization gradually appears during mouse oocyte growth. Oocytes at late stages show self-interacting, cohesin-independent compartmental domains marked by H3K27me3, therefore termed Polycomb-associating domains (PADs). PADs and inter-PAD (iPAD) regions form compartment-like structures with strong inter-domain interactions among nearby PADs. PADs disassemble upon meiotic resumption from diplotene arrest but briefly reappear on the maternal genome after fertilization. Upon maternal depletion of Eed, PADs are largely intact in oocytes, but their reestablishment after fertilization is compromised. By contrast, depletion of Polycomb repressive complex 1 (PRC1) proteins attenuates PADs in oocytes, which is associated with substantial gene de-repression in PADs. These data reveal a critical role of Polycomb in regulating chromatin architecture during mammalian oocyte growth and early development.

Imprinted X-chromosome inactivation impacts primitive endoderm differentiation in mouse blastocysts.

Epigenetic and transcriptome alterations are essential for lineage specification, represented by imprinted X-chromosome inactivation (iXCI) in female mouse preimplantation embryos. However, how various factors affect transcriptome states and lineage commitment remains unclear. We found that in vitro culture duration strongly influences transcriptional variation compared to iXCI loss. Single-cell analysis of the inner cell mass (ICM) for major transcription and epigenomic factors revealed that sex-specific differences in expression are diminished by loss of iXCI in the primitive endoderm (PrE) but not in the epiblast. Females had a higher proportion of ICM compared to that in males, and PrE development was affected by iXCI states in female embryos. Our findings provide insight into sex differences and iXCI function in lineage specification.

Intranuclear characteristics of pig oocytes stained with brilliant cresyl blue and nucleologenesis of resulting embryos.

Brilliant cresyl blue (BCB) vital labelling is a powerful method for analyzing the quality of porcine cumulus-oocyte complexes. Our aim was to investigate the correlation between the selection of porcine oocytes using BCB labelling and selected intranuclear characteristics of porcine oocytes and parthenotes. Moreover, BCB labelling was correlated with the diameter of the oocyte and the developmental potential of the parthenotes. The following methods were used: BCB labelling, measurement of the diameter of the oocyte, parthenogenetic activation, immunocytochemistry, transmission electron microscopy, enucleation and relative protein concentration (RPC) analysis. We determined that the diameter of the oocytes in the BCB-positive (BCB+) group was significantly larger than in the BCB-negative (BCB-) group. Immediately after oocyte selection according to BCB labelling, we found significant difference in chromatin configuration between the analyzed groups. BCB+ oocytes were significantly better at maturation than BCB- oocytes. BCB+ embryos were significantly more competent at cleaving and in their ability to reach the blastocyst stage than BCB- embryos. Ultrastructural analyses showed that the formation of active nucleoli in the BCB+ group started at the 8-cell stage. Conversely, most BCB- embryos at the 8-cell and 16-cell stages were fragmented. No statistically significant difference in RPC in nucleolus precursor bodies (NPBs) between BCB+ and BCB- oocytes was found. We can conclude that BCB labelling could be suitable for assessing the quality of porcine oocytes. Moreover, the evaluation of RPC indicates that the quantitative content of proteins in NPB is already established in growing oocytes.

Non-invasive mitochondrial DNA quantification on Day 3 predicts blastocyst development: a prospective, blinded, multi-centric study.

In ART, embryo quality evaluation is routinely based on morphological criteria. We previously demonstrated that the mitochondrial DNA (mtDNA)/genomic DNA (gDNA) ratio in culture medium was significantly associated with embryo quality and viability potential. The purpose of this prospective, blinded, multi-centric study was to validate the use of mtDNA/gDNA ratio in Day 3 spent medium as a predictor of human embryo developmental competence. The mtDNA/gDNA ratio was assessed in Day 3 culture media (n=484) of embryos from 143 patients by quantitative PCR. A mixed effect logistic regression model was applied. We found that mtDNA/gDNA ratio in Day 3 culture medium combined with embryo morphology improves the prediction upon blastulation compared to morphology alone (P < 0.0001), independent of patient and cycle characteristics. With regard to routine use in clinics, we evaluated the ability of the novel, combined grading score to improve selection of developmentally competent embryos of a single cohort. Including embryos from 44 patients, the sensibility and specificity of the scoring system based on Day 3 morphological stage were 92% and 13%, respectively. Integration with the culture medium mtDNA/gDNA ratio increased the performance of the method (sensibility: 95%; specificity: 65%). The results of this study suggest the possibility of carrying out a non-invasive evaluation of embryonic mtDNA content through the culture medium. When combined with embryo morphology, it has the potential to help embryologists rank embryos and choose which embryo(s) has the greater development potential, and thus should be transferred on Day 3, among sibling embryos with the same morphological grade.

Clinical application of embryo aneuploidy testing by next-generation sequencing.

We review here the evolution in the field of embryo aneuploidy testing over the last 20 years, from the analysis of a subset of chromosomes by fluorescence in situ hybridisation to the transition toward a more comprehensive analysis of all 24 chromosomes. This current comprehensive aneuploidy testing most commonly employs next-generation sequencing (NGS). We present our experience in over 130 000 embryo biopsies using this technology. The incidence of aneuploidy was lower in trophectoderm biopsies compared to cleavage-stage biopsies. We also confirmed by NGS that embryo aneuploidy rates increased with increasing maternal age, mostly attributable to an increase in complex aneuploid embryos. In contrast, the number of MII oocytes retrieved or the use of oocyte vitrification did not affect aneuploidy rates. Similarly, neither maternal age, oocyte number, nor oocyte vitrification affected the incidence of mosaicism. Analysis of clinical outcomes, indications, and potential benefits of embryo aneuploidy testing revealed advanced maternal age as the most favored group, with some evidence of improved delivery rate per transfer as well as decreased miscarriage rates and time to pregnancy. Other indications are: recurrent miscarriage, repetitive implantation failure, severe male factor, previous trisomic pregnancy, and good prognosis patients mainly undergoing single embryo transfer, with the latter indication used to reduce the occurrence of multiple pregnancies without compromising cycle outcome. In conclusion, NGS has become the most appropriate technology for aneuploidy testing in trophectoderm biopsies, with accurate results, high throughput, and cost efficiency. This technology can be also applied to the analysis of the embryonic cell free DNA released to the culture media at blastocyst stage. This is a promising approach towards a non-invasive preimplantation genetic testing of aneuploidy.

Influence of follicle size on bovine oocyte lipid composition, follicular metabolic and stress markers, embryo development and blastocyst lipid content.

This study assessed the lipid composition of oocytes from different follicle sizes and compared the expression of lipid-related genes and follicular fluid (FF) molecules between groups. We also investigated the functional consequences of differences on embryo development and blastocyst lipid deposits. Oocytes and FF were recovered from different follicle sizes. Oocytes from small (≤5mm) and large (≥6mm) bovine follicles were used to produce Day 7 expanded blastocysts (Day7Ex) and blastocysts that only became expanded at Day 8 (Day8Ex) after insemination. Oocytes from >8mm follicles had the highest lipid content. Few oocyte phospholipid variations were identified between groups. Very long chain fatty acid elongase 6 (ELOVL6) mRNA abundance was reduced in larger follicle-derived oocytes compared with the ≤2mm group. Increased levels of glucose, reactive oxygen species, glutathione and superoxide dismutase activity were also identified in FF from larger follicles. Large follicle-derived embryo development and lipid content of Day7Ex were greater than those derived from small follicles. Day8Ex had greater lipid deposition than Day7Ex. Oocytes and blastocysts exhibited follicle size-specific lipids. Large-follicle oocytes had increased lipid content and became Day7Ex with greater lipid deposition whereas delayed blastocoel expansion associated with a prolonged period of culture determined the lipid accumulation of Day8Ex. The FF microenvironment of large follicles seems to favour embryo development.

Simultaneous RNA-DNA FISH in Mouse Preimplantation Embryos.

Fluorescent in situ hybridization (FISH) is a powerful cytogenetic technique that allows the visualization and quantification of RNA and DNA molecules in different cellular contexts. In general, FISH applications help to advance research, cytogenetics, and diagnostics. DNA FISH can be applied, for example, for gene mapping and for detecting genetic aberrations. RNA FISH provides information about gene expression. However, in cases where RNA and DNA molecules need to be detected in the same sample, the result is often compromised by the fact that the tissue sample is damaged due to the multitude of processing steps that are required for each application. In addition, the sequential application of RNA and DNA FISH protocols on the same sample is very time consuming. Here we describe a brief protocol that enables the combined and simultaneous detection of Xist RNA and centromeric DNA of chromosome 6 in mouse preimplantation embryos. In addition, we describe how to generate indirect-labeled probes starting from BACs. This protocol may be applied to any combination of RNA and DNA detection.

Combined Immunofluorescence, RNA FISH, and DNA FISH in Preimplantation Mouse Embryos.

Transcriptional and epigenetic dynamics of the genome occur during early development in mammals. It has been difficult to study these dynamics due to the limitation of materials and the difficulty of handling. In this chapter, we describe our attempt to apply a combination of immunofluorescence (IF), and RNA and DNA fluorescent in situ hybridization (FISH) in preimplantation mouse embryos. We have concentrated on refining these techniques to study the dynamics of X chromosome inactivation in mouse embryos. The techniques and general underlying principles described here should be applicable to other mammals of interest.