Blastomeres Of Embryos Research Articles

Klf5 establishes bi-potential cell fate by dual regulation of ICM and TE specification genes.

SUMMARYEarly blastomeres of mouse preimplantation embryos exhibit bi-potential cell fate, capable of generating both embryonic and extra-embryonic lineages in blastocysts. Here we identify three major two-cell-stage (2C)-specific endogenous retroviruses (ERVs) as the molecular hallmark of this bi-potential plasticity. Using the long terminal repeats (LTRs) of all three 2C-specific ERVs, we identify Krüppel-like factor 5 (Klf5) as their major upstream regulator. Klf5 is essential for bi-potential cell fate; a single Klf5-overexpressing embryonic stem cell (ESC) generates terminally differentiated embryonic and extra-embryonic lineages in chimeric embryos, and Klf5 directly induces inner cell mass (ICM) and trophectoderm (TE) specification genes. Intriguingly, Klf5 and Klf4 act redundantly during ICM specification, whereas Klf5 deficiency alone impairs TE specification. Klf5 is regulated by multiple 2C-specific transcription factors, particularly Dux, and the Dux/Klf5 axis is evolutionarily conserved. The 2C-specific transcription program converges on Klf5 to establish bi-potential cell fate, enabling a cell state with dual activation of ICM and TE genes.

Asymmetric expression of maternal mRNA governs first cell-fate decision.

The goal of preimplantation development is to establish the fates of the embryonic and extra-embryonic cells. However, when and how cell fates are determined during early mammalian embryonic development remains unclear. We report that the high mobility group (HMG) protein family member HMGA1 was distributed differentially in mouse two-cell blastomeres. Knockdown of Hmga1 expression in one of the two cells reduced the number of cells contributing to the inner cell mass (ICM), suggesting that differential distribution of HMGA1 in the blastomeres in two-cell mouse embryos affected the selection of embryonic cell lineages. Mechanistically, HMGA1 promotes the expression of the ICM-specific gene Sox2. The results of this study show that mouse embryos demonstrate heterogeneity as early as the two-cell stage, and that these differences are related to cell-fate differentiation in early mouse embryos.

Number of Blastomeres in Day-2 Embryos Affect the Rates of Blastocyst Formation and Clinical Pregnancy During In Vitro Fertilization Cycles.

In the current in vitro fertilization and embryo transfer protocol, the 8 blastomeres in the day-3 embryo are selected for transfer because these embryos can produce high rates of blastocyst formation and clinical pregnancy. However, the relationship between the blastomere number in day-2 embryos and the rate of blastocyst formation or clinical pregnancy remains unclear. The purpose of this retrospective study is to explore the relationship between the blastomere number in day-2 embryos and the rate of blastocyst formation or clinical pregnancy. From January 2015 to April 2020, we collected 8126day-3 embryos (8 blastomeres) from 2282 patients. These embryos were classified into 8 groups (1 blastomere, 2 blastomeres, 3 blastomeres, 4 blastomeres, 5 blastomeres, 6 blastomeres, 7 blastomeres, and 8 blastomeres) based on their blastomeres number on day 2 after insemination. Of these groups, the 4 blastomeres group accounted for the largest proportion (74.44%). The 1 blastomere group accounted for the smallest proportion (0.22%). A total of 3554day-3 embryos (8 blastomeres) from 1648 patients developed into blastocysts. The rate of blastocyst formation from the 4 blastomeres group was the highest (94.06%). Finally, 800 patients received single day-3 embryos (8 blastomeres) transfer. The rate of clinical pregnancy from 4 blastomeres group was the highest (51.98%). In conclusion, our data provide evidence that the number of blastomeres in day-2 embryos affects the rate of blastocyst formation and clinical pregnancy.

Dual spindles assemble in bovine zygotes despite the presence of paternal centrosomes.

The first mitosis of the mammalian embryo must partition the parental genomes contained in two pronuclei. In rodent zygotes, sperm centrosomes are degraded, and instead, acentriolar microtubule organizing centers and microtubule self-organization guide the assembly of two separate spindles around the genomes. In nonrodent mammals, including human or bovine, centrosomes are inherited from the sperm and have been widely assumed to be active. Whether nonrodent zygotes assemble a single centrosomal spindle around both genomes or follow the dual spindle self-assembly pathway is unclear. To address this, we investigated spindle assembly in bovine zygotes by systematic immunofluorescence and real-time light-sheet microscopy. We show that two independent spindles form despite the presence of centrosomes, which had little effect on spindle structure and were only loosely connected to the two spindles. We conclude that the dual spindle assembly pathway is conserved in nonrodent mammals. This could explain whole parental genome loss frequently observed in blastomeres of human IVF embryos.

P–224 Multinucleated and non-multinucleated embryos in PGT-A cycles: Is there any difference?

Abstract Study question Is multinucleation during cleavage stage correlated with the ploidy status of embryos and how does it affect the clinical outcome? Summary answer The presence of multinucleated embryos does not affect clinical outcome, although the risk of aneuploidy is higher in multinucleated embryos. What is known already Multinucleated blastomeres (ΜΝ) of cleavage stage embryos has been reported widely in scientific literature. Multinucleation has been associated with diminished embryo developmental competency and clinical outcomes such as lower implantation. Although this is an intriguing subject of research, it is not clear yet whether multinucleation is related to aneuploidy. Morphological irregularities such as multinucleation in blastomeres became a constant finding only after the perpetually evolving technology of time-lapse culture of embryos which in combination with PGT-A analysis, creates new research paths which aim to develop a new tool for selection or deselection of embryos for transfer. Study design, size, duration This retrospective study, included 97 PGT-A cycles, performed at Embryolab fertility clinic from May 2017 to December 2020, all cultured in time-lapse incubator (EmbryoScope). Two study groups were formed; the MN Group consisted of PGT-A cycles with at least one multinucleated embryo (n = 56) and the Control Group in which all PGT-A cycles had no multinucleated embryos (n = 38). Euploidy rate, type of chromosomal abnormality, cumulative pregnancy and live birth rates were compared between the two groups. Participants/materials, setting, methods Embryos were annotated for the existence of multinucleated blastomeres on Day 2 of their development. Biopsy was performed on Days 5/6 and embryos were genetically tested. One or two euploid embryos were transferred. Euploidy rate and clinical outcomes between the two groups were compared. Within the MN group, euploidy rate between multinucleated and non-multinucleated embryos was compared. For abnormal embryos, association of multinucleation with the type of abnormality was tested. SAS statistical analysis was performed. Main results and the role of chance Mean female age was 35.93 years in the MN group and 38.39 years in the control group. Blastocyst formation rate (expressed per fertilised oocytes) was similar between MN and Control group (74% vs 76%, p = 0.6303). In the MN group, 56 cases resulted in 44 embryo transfers while in the control group 38 cases resulted in 23 embryo transfers. Pregnancy rates (59.09% vs 65.21%, p = 0.6255) and clinical pregnancy rates (45.45% vs 39.13%, p = 0.4245) were not significantly different between MN and Control group. Initially, cumulative live birth rate was found to be significantly higher in the MN group compared to the Control group (62.96% vs 33.34%, p = 0.0417). However, when logistic and poisson regression was applied, it became obvious that this difference was not affected by multinucleation but from other factors such as female age. When comparing multinucleated and non-multinucleated embryos within the MN group, it was found that the mean number of euploid embryos was significantly higher in the non-multinucleated subgroup of embryos (p = 0.0021). No correlation was found between multinucleation and the type of chromosomal abnormality. Limitations, reasons for caution The sample size is the main limitation of the present study. More research with bigger sample size is needed in order to confirm the finding of the present study. Wider implications of the findings: The present study suggests that multinucleated blastomeres during embryo development is not an indication for diminished blastocyst formation and does not affect the clinical outcomes. However, within a sibling embryo population, non-multinucleated embryos tend to be euploid and this finding can be used to advance embryo selection efficiency. Trial registration number Not applicable

Genotoxic Effects of Radiofrequency-Electromagnetic Fields

Genotoxic Effects of Radiofrequency-Electromagnetic Fields

Totipotency of mouse zygotes extends to single blastomeres of embryos at the four-cell stage

In multicellular organisms, oocytes and sperm undergo fusion during fertilization and the resulting zygote gives rise to a new individual. The ability of zygotes to produce a fully formed individual from a single cell when placed in a supportive environment is known as totipotency. Given that totipotent cells are the source of all multicellular organisms, a better understanding of totipotency may have a wide-ranging impact on biology. The precise delineation of totipotent cells in mammals has remained elusive, however, although zygotes and single blastomeres of embryos at the two-cell stage have been thought to be the only totipotent cells in mice. We now show that a single blastomere of two- or four-cell mouse embryos can give rise to a fertile adult when placed in a uterus, even though blastomere isolation disturbs the transcriptome of derived embryos. Single blastomeres isolated from embryos at the eight-cell or morula stages and cultured in vitro manifested pronounced defects in the formation of epiblast and primitive endoderm by the inner cell mass and in the development of blastocysts, respectively. Our results thus indicate that totipotency of mouse zygotes extends to single blastomeres of embryos at the four-cell stage.

Extranuclear Inheritance of Mitochondrial Genome and Epigenetic Reprogrammability of Chromosomal Telomeres in Somatic Cell Cloning of Mammals.

The effectiveness of somatic cell nuclear transfer (SCNT) in mammals seems to be still characterized by the disappointingly low rates of cloned embryos, fetuses, and progeny generated. These rates are measured in relation to the numbers of nuclear-transferred oocytes and can vary depending on the technique applied to the reconstruction of enucleated oocytes. The SCNT efficiency is also largely affected by the capability of donor nuclei to be epigenetically reprogrammed in a cytoplasm of reconstructed oocytes. The epigenetic reprogrammability of donor nuclei in SCNT-derived embryos appears to be biased, to a great extent, by the extranuclear (cytoplasmic) inheritance of mitochondrial DNA (mtDNA) fractions originating from donor cells. A high frequency of mtDNA heteroplasmy occurrence can lead to disturbances in the intergenomic crosstalk between mitochondrial and nuclear compartments during the early embryogenesis of SCNT-derived embryos. These disturbances can give rise to incorrect and incomplete epigenetic reprogramming of donor nuclei in mammalian cloned embryos. The dwindling reprogrammability of donor nuclei in the blastomeres of SCNT-derived embryos can also be impacted by impaired epigenetic rearrangements within terminal ends of donor cell-descended chromosomes (i.e., telomeres). Therefore, dysfunctions in epigenetic reprogramming of donor nuclei can contribute to the enhanced attrition of telomeres. This accelerates the processes of epigenomic aging and replicative senescence in the cells forming various tissues and organs of cloned fetuses and progeny. For all the above-mentioned reasons, the current paper aims to overview the state of the art in not only molecular mechanisms underlying intergenomic communication between nuclear and mtDNA molecules in cloned embryos but also intrinsic determinants affecting unfaithful epigenetic reprogrammability of telomeres. The latter is related to their abrasion within somatic cell-inherited chromosomes.

Novel technique for definite blastomere inhibition and distribution of maternal RNA in sterlet Acipenser ruthenus embryo

The cleavage pattern of a vertebrate’s embryo is either holoblastic (complete) or meroblastic (partial). Sturgeon and other basal bony fishes represent a transition of the cleavage pattern. To understand the transition, it is essential to develop an effective technique for the inhibition of specific blastomere cleavage. So far, various studies have demonstrated that diatom-derived polyunsaturated aldehyde (PUA), 2,4-decadienal (DD)—a model aldehyde for experimental studies—adversely affects the developing embryos of several aquatic species. In this study, we employed DD for inhibition of cleavage of a definite blastomere in sturgeon embryos under various conditions. The effective treatment was found to be a combination of DD injection (0.01 v/v) and visible light (44.86–91.15 W m−2). Notably, DD injection or light irradiation alone cannot inhibit cleavage. Furthermore, spatial RNA localization analysis using quantitative polymerase chain reaction (qPCR)-tomography revealed that the localized pattern of selected maternal messenger ribonucleic acids (mRNAs) remained constant along the animal–vegetal (A-V) axis, which suggests that RNA localization is completed by the end of oogenesis and that early embryonic cleavage is not required for A-V asymmetry preservation.

Automated three-dimensional image processing for 2-blastomere and 4-blastomere embryo surgical applications

The surgical tool and embryo blastomere locations are crucial to perform cell surgery. This paper presents an automated algorithm to compute blastomere centroids and surgical tool coordinate. The algorithm utilises the camera to procure Z-stack images of the workspace. It uses a three-dimensional (3D) canny filter and k-means clustering to segment and compute 3D centroid of each blastomere. For surgical tool tip location, region of interest (ROI) is established via template matching. 2D canny filter is applied on the ROI to produce segmentation results. These results are compiled together to estimate the 3D tool tip location. All computations are conducted on image plane which provide convenience to implement image-based visual servoing (IBVS) control in the future. With the overall obtained computation variations for 2-blastomere (2B) and 4-blastomere (4B) embryo, the proposed algorithm is suitable for blastomere aspiration alike procedures.

Development of PCR based approach to detect potential mosaicism in porcine embryos

Direct injection of genome editing tools such as CRISPR/Cas9 system into developing embryos has been widely used to generate genetically engineered pigs. The approach allows us to produce pigs carrying targeted modifications at high efficiency without having to apply somatic cell nuclear transfer. However, the targeted modifications during embryogenesis often result in mosaicism, which causes issues in phenotyping founder animals and establishing a group of pigs carrying intended modifications. This study was aimed to establish a genomic PCR and sequencing system of a single blastomere in the four-cell embryos to detect potential mosaicism. We performed genomic PCR in four individual blastomeres from four-cell embryos. We successfully amplified target genomic region from single blastomeres of 4-cell stage embryo by PCR. Sanger sequencing of the PCR amplicons obtained from the blastomeres suggested that PCR-based genotyping of single blastomere was a feasible method to determine mutation type generated by genome editing technology such as CRISPR/Cas9 in early stage embryos. In conclusion, we successfully genotyped single blastomeres in a single 4-cell stage embryo to detect potential mosaicism in porcine embryos. Our approach offers a simple platform that can be used to screen the prevalence of mosaicism from designed CRISPR/Cas9 systems.

Protein regulator of cytokinesis 1 regulates chromosome dynamics and cytoplasmic division during mouse oocyte meiotic maturation and early embryonic development.

In contrast to the homeokinesis of mitosis, asymmetric division of cytoplasm is the conspicuous feature of meiosis in mammalian oocytes. Protein regulator of cytokinesis 1 (PRC1) is an important regulator during mitotic spindle assembly and cytoplasmic division, but its functions in oocyte meiosis and early embryo development have not been fully elucidated. In this study, we detected PRC1 expression and localization and revealed a nuclear, spindle midzone-related dynamic pattern throughout meiotic and mitotic progressions. Treatment of oocytes with the reagents taxol or nocodazole disturbed the distribution of PRC1 in metaphase II oocytes. Further, PRC1 depletion led to failure of first polar body (PB1) extrusion and spindle migration, aneuploidy and defective kinetochore-microtubule attachment and spindle assembly. Overexpression of PRC1 resulted in PB1 extrusion failure, aneuploidy and serious defects of spindle assembly. To investigate PRC1 function in early embryos, we injected Prc1 morpholino into zygotes and 2-cell stage embryos. Depletion of PRC1 in zygotes impaired 4-cell, morula and blastocyst formation. Loss of PRC1 in single or double blastomeres in 2-cell stage embryos significantly impaired cell division, indicating its indispensable role in early embryo development. Co-immunoprecipitation showed that PRC1 interacts with polo-like kinase 1 (PLK1), and functional knockdown and rescue experiments demonstrated that PRC1 recruits PLK1 to the spindle midzone to regulate cytoplasmic division during meiosis. Finally, kinesin family member 4 knockdown downregulates PRC1 expression and leads to PRC1 localization failure. Taken together, our data suggest PRC1 plays an important role during oocyte maturation and early embryonic development by regulating chromosome dynamics and cytoplasmic division.

A New Day 4 Grading System to Assess Embryo Quality in Frozen Embryo Transfer Cycles

To present a new day 4 (D4) embryo grading system to assess embryos in frozen-thawed embryo transfer (FET) cycles. A new grading system (grades A-E) was developed from the 2011 ESHRE Istanbul Consensus for D4 embryos in FET cycles. Embryos with complete compaction were classified as grade A; those with partial compaction were assigned as grade B; and those without compaction were classified as grades C, D, and E according to their different blastomere number ratio (BNR; number of embryo blastomeres on D4/number of embryo blastomeres on D3, D4/D3). Embryos with a BNR of ≥ 1.5 were defined as grade C, those with a BNR of ≥ 1.2 and < 1.5 were defined as grade D, and those with a BNR of ≥ 1.0 and < 1.2 were defined as grade E. Using this proposed grading model, 5460 embryos with known implantation data were retrospectively analyzed after D4 transfer in FET cycles. The transferred embryos exhibited a similar declining trend in implantation and live birth rates from the top grade A to the lowest grade E. The in vitro fertilization group showed increased implantation rates of grade B and E embryos compared with the intracytoplasmic sperm injection group (grade B: 41.99%, 34.63%, χ2 = 5.84, p < 0.05 and grade E: 18.98%, 14.08, χ2 = 75.62, p < 0.01). Receiver-operating characteristic analysis revealed that our proposed model predicted the implantation outcomes and live birth rates of all embryos (area under the curve = 0.65; 95% confidence interval [CI],0.63-0.66; p < 0.01 and AUC = 0.73; 95%CI, 0.65-0.84; p < 0.001, respectively). This study demonstrates that the new grading system provided by us can be a useful tool for assisting embryo selection via changes in embryo morphology. D4 embryo transfer provides a simple and applicable method for FET cycles in daily practice.

NEDD4-like ubiquitin ligase 2 protein (NEDL2) in porcine spermatozoa, oocytes, and preimplantation embryos and its role in oocyte fertilization†.

The ubiquitin-proteasome system plays diverse regulatory and homeostatic roles in mammalian reproduction. Ubiquitin ligases are the substrate-specific mediators of ubiquitin-binding to its substrate proteins. The NEDD4-like ubiquitin ligase 2 (aliases NEDL2, HECW2) is a HECT-type ubiquitin ligase that contains one N-terminal HECW ubiquitin ligase domain, one C-terminal HECT ubiquitin ligase domain, one C2 domain, and two WW protein-protein interaction modules. Beyond its predicted ubiquitin-ligase activity, its cellular functions are largely unknown. Current studies were designed to investigate the content and distribution of NEDL2 in porcine spermatozoa, oocytes, zygotes, and early preimplantation embryos, and in cumulus cells before and after in vitro maturation with oocytes, and fibroblast cells as positive control by western blot and immunocytochemistry, and to examine its roles during oocyte fertilization. Multiple isoforms of NEDL2 were identified by WB. One at approximately 52kDa was detected only in the germinal vesicle (GV) stage and metaphase II oocytes, and in early preimplantation embryos. Other isoforms were high mass bands at 91, 136, and 155kDa, which were only detected in somatic cells. Interestingly, ejaculated spermatozoa prominently displayed the same 52kDa band as oocytes; they also had two minor bands of 74 and 129kDa, which were not detected in somatic cells or oocytes. By immunofluorescence, NEDL2 showed a diffused cytoplasmic localization in all cell types and accumulated in distinct foci in the germinal vesicles (GVs) of immature oocytes, in maternal and paternal pronuclei of zygotes and nuclei of embryo blastomeres and somatic cells. In blastocysts, the labeling intensity of NEDL2 was stronger in the inner cell mass than in trophoblast, indicating higher NEDL2 content in the ICM cells than in trophectoderm. NEDL2 abundance was 10 times higher in post-maturation oocyte-surrounding cumulus cells than that of cumulus cells before in vitro maturation with hormones, indicating that NEDL2 may have a unique role in cumulus cells after ovulation. Microinjection of anti-NEDL2 antibody into oocyte before IVF did not affect the percentage of oocytes fertilized, percentage of oocytes cleaved, or blastocyst formation. However, the anti-NEDL2 antibody decreased the number of pronuclei, accelerated the formation of nuclear precursor bodies at 6h postfertilization, inhibited sperm DNA decondensation, and resulted in more fertilized oocytes without male pronuclear formation. In summary, NEDL2 may play a key role during fertilization, especially during sperm DNA decondensation.

Distribution of introduced human mitochondrial DNA in early stage mouse embryos

Objective. The aim of study was the analysis of human mitochondrial DNA (mtDNA) distribution among murine blastomeres in the embryos developing after an injection of human mitochondria suspension at the stage of one or two cells is presented.&#x0D; Material and methods. Mice CBA/C57Black from Rappolovo aged three weeks were used. Zygotes were obtained upon hormonal stimulation of animals and mated with males. 310 pL of mitochondrial suspension from HepG2 cells was injected into a zygote or one blastomere of a two-cell embryo. Zygotes or two-cell embryos cultured in M3 medium drops covered with mineral oil in Petri dishes. Upon reaching the two-, four- or eight-cell stage the cultured embryos were separated into blastomeres. The latter were lysed and the total DNA was isolated. Human mtDNA was detected by PCR using species-specific primers.&#x0D; Results. The development of 2848 mouse embryos was monitored. In 520 embryos that achieved the stage of 2, 4, 8 in proper time the presence of human mtDNA was assayed in each blastomere. Along with murine mtDNA all embryos contained human mitochondrial genome, which is an evidence of artificially modelled heteroplasmy. Not every blastomere of transmitochondrial embryos contained foreign (human) mtDNA. Mathematical elaboration evidenced an uneven distribution of human mtDNA in cytoplasm within the time elapsed between the injection of human mitochondria and the subsequent splitting of the embryo.&#x0D; Conclusion. The results obtained confirm our previous notion of the presence of 1011 segregation units of human mtDNA in the total amount of mitochondria (about 5 ∙ 102) injected into an embryo.

The molecular and cellular features of 2-cell-like cells: a reference guide.

Currently, two main cell culture models predominate pluripotent stem cell research: embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). Thanks to their ability to contribute to and form all tissues within the body, ESCs and iPSCs have proven invaluable in understanding pluripotent states, early embryonic development and cell differentiation, as well as in devising strategies for regenerative medicine. Comparatively little is known about totipotency - a cellular state with greater developmental potential. In mice, only the zygote and the blastomeres of the 2-cell-stage embryo are truly totipotent, as they alone can develop to form the embryo and all of its supportive extra-embryonic tissues. However, the discovery of a rare subpopulation of cells in murine ESC cultures, possessing features of 2-cell embryo blastomeres and expanded cell fate potential, has provided a biochemically tractable model to enable the in vitro study of totipotency. Here, we summarize current known features of these 2-cell-like cells (2CLCs) in an effort to provide a reference for the community, and to clarify what we know about their identity so far.

GOTI, a method to identify genome-wide off-target effects of genome editing in mouse embryos.

Genome editing holds great potential for correcting pathogenic mutations. We developed a method called GOTI (genome-wide off-target analysis by two-cell embryo injection) to detect off-target mutations by editing one blastomere of two-cell mouse embryos using either CRISPR-Cas9 or base editors. GOTI directly compares edited and non-edited cells without the interference of genetic background and thus could detect potential off-target variants with high sensitivity. Notably, the GOTI method was designed to detect potential off-target variants of any genome editing tools by the combination of experimental and computational approaches, which is critical for accurate evaluation of the safety of genome editing tools. Here we provide a detailed protocol for GOTI, including mice mating, two-cell embryo injection, embryonic day 14.5 embryo digestion, fluorescence-activated cell sorting, whole-genome sequencing and data analysis. To enhance the utility of GOTI, we also include a computational workflow called GOTI-seq (https://github.com/sydaileen/GOTI-seq) for the sequencing data analysis, which can generate the final genome-wide off-target variants from raw sequencing data directly. The protocol typically takes 20 d from the mice mating to sequencing and 7 d for sequencing data analysis.

Inverse relationship between autophagy and CTSK is related to bovine embryo quality.

Improving the quality and the developmental competence of in vitro produced (IVP) embryos is an indispensable goal for assisted reproductive technology. Autophagy is a major protective mechanism for intracellular degradation of unnecessary cytoplasmic components. Autophagy ends by the fusion between autophagic vacuoles and lysosomes, allowing the degradation of the cargo by lysosomal enzymes, especially the cathepsins (CTSs). However, it is still unclear how autophagy and cathepsin K (CTSK) relate to embryo development. This study evaluated (1.) the activities of autophagy and CTSK in relation to bovine embryo quality and (2.) the effect of autophagy induction and/or CTSK inhibition on preimplantation embryo development and quality. We show here that good-quality embryos exhibited a greater autophagic activity and less CTSK activity compared to poor-quality embryos. Blastomeres of an individual embryo may vary in their quality. Good quality blastomeres showed an increased autophagic activity and decreased CTSK activity compared to poor-quality blastomeres within the same embryo at different developmental stages. Importantly, induction of autophagy and/or inhibition of CTSK improved the developmental rate (increased blastocyst and hatching rates) and the quality (increased total cell number and decreased the percentage of apoptotic cells) of IVP bovine embryos. These results demonstrate a promising approach to selectively isolate good-quality embryos and improve the efficiency of IVEP of cattle embryos.

Next-generation sequencing analysis of each blastomere in good-quality embryos: insights into the origins and mechanisms of embryonic aneuploidy in cleavage-stage embryos.

To explore the whole-chromosome status, origins, and mechanisms of chromosomal abnormalities in good-quality cleavage embryos using multiple annealing and looping-based amplification cycle (MALBAC) sequencing. The embryos studied came from7 patients (maternal aged 26-35) who had healthy birth from the same IVF cycles. These 21 frozen day 3 good-quality embryos were thawed and disaggregated into individual blastomere. Each blastomere was collected and analyzed by MALBAC sequencing. Conclusive results were obtained from a high percentage of blastomeres (95.3%). A total of 46.6% of blastomeres were diploid, 53.4% were abnormal, and 28.0% had complex aneuploidy. Out of 21 embryos, 3 (14.3%) were normal and 18 (85.7%) were mosaics, showing the occurrence of mitotic errors; aneuploidy was confirmed in all cells of 4 of the 18 embryos, which showed the coexistence of meiotic errors. Conclusive results were obtained from all blastomeres of 15 embryos (71.4%, 15/21), which enabled us to reconstruct the cell lineage on the basis of the chromosomal content of the blastomeres in each division. There were 9 mitotic errors (8.7%, 9/103): nondisjunction accounted for 88.9% (8/9), and endoreplication accounted for 11.1% (1/9). In good-quality embryos, there was a high rate and diverse array of chromosomal abnormalities. Morphological evaluation does not appear to assist in the reduction in meiotic errors from parental origins. Mitotic errors were common, and nondisjunction was found to be the main mechanism causing malsegregation during the cleavage divisions.

Maternally contributed Nlrp9b expressed in human and mouse ovarian follicles contributes to early murine preimplantation development.

The aim of the study is to investigate presence and role of the gene encoding the maternally contributed nucleotide-binding oligomerization domain (NOD)-like receptors with a pyrin domain (PYD)-containing protein 9 (NLRP9) in human and mouse ovaries, respectively, and in preimplantation mouse embryo development by knocking down Nlrp9b. Expression levels of NLRP9 mRNA in human follicles were extracted from RNA sequencing data from previous studies. In this study, we performed a qPCR analysis of Nlpr9b mRNA in mouse oocytes and found it present. Intracellular ovarian distribution of NLRP9B protein was accomplished using immunohistochemistry. The distribution of NLRP9B was explored using a reporter gene approach, fusing NLRP9B to green fluorescent protein and microinjection of in vitro-generated mRNA. Nlrp9b mRNA function was knocked down by microinjection of short interference (si) RNA targeting Nlrp9b, into mouse pronuclear zygotes. Knockdown of the Nlrp9b mRNA transcript was confirmed by qPCR. We found that the human NLRP9 gene and its corresponding protein are highly expressed in human primordial and primary follicles. The NLRP9B protein is localized to the cytoplasm in the blastomeres of a 2-cell embryo in mice. SiRNA-mediated knockdown of Nlrp9b caused rapid elimination of endogenous Nlrp9b mRNA and premature embryo arrest at the 2- to 4-cell stages compared with that of the siRNA-scrambled control group. These results suggest that mouse Nlrp9b, as a maternal effect gene, could contribute to mouse preimplantation embryo development. It remains to investigate whether NLRP9 have a crucial role in human preimplantation embryo and infertility.