An approach for live imaging of first cleavage in mouse embryos using fluorescent chemical probes for DNA, microtubules, and microfilaments.

Purpose: Dynamic morphological changes in the chromosome and cytoskeleton occur in mammals and humans during early embryonic development, and abnormalities such as embryonic chromosomal aneuploidy occur when development does not proceed normally. Visualization of the intracellular organelles and cytoskeleton allows elucidation of the development of early mammalian embryos. The behavior of the DNA and cytoskeleton in early mammalian embryos has conventionally been observed by injecting target molecule mRNAs, incorporating a fluorescent substance-expressing gene, into embryos. In this study, we visualized the chronological behavior of male and female chromosome condensation in mouse embryos, beginning in the two-pronuclear zygote, through the first division to the two-cell stage, using fluorescent chemical probes to visualize the behavior of DNA, microtubules, and microfilaments. Method: Mouse two-pronuclear stage embryo were immersed in medium containing fluorescent chemical probes to visualize DNA, microtubules, and microfilaments. Observation was performed with a confocal microscope. Results: This method allowed us to observe how chromosome segregation errors in first somatic cell divisions in mouse embryos and enabled dynamic analysis of a phenomenon called lagging chromosomes. Conclusions: By applying this method, we can observe any stage of embryonic development, which may provide new insights into embryonic development in other mammals.

Consistency between chromosomal status analysis of biopsied human blastocyst trophectoderm cells and whole blastocyst cells.

PURPOSE: This study investigated the consistency between results of preimplantation genetic testing for aneuploidy performed on trophectoderm (TE) cells and remaining blastocyst cells. METHODS: TE biopsy was performed on 29 surplus cryopreserved human blastocysts. Biopsy samples and remaining blastocysts were processed using the VeriSeq PGS kit, and chromosomal statuses were compared by next-generation sequencing. RESULTS: Discordance was observed in the chromosomal status of 11 out of 29 blastocysts between the biopsied TE and remaining blastocysts. Concordance was observed in 11 of 12 blastocysts classified as euploid by TE biopsy and in 7 of 17 blastocysts classified as aneuploid. There was 100% concordance (7/7) in cases diagnosed as aneuploid with no mosaicism by TE biopsy. However, discordance was observed in all 10 cases showing mosaicism or partial chromosomal abnormality. CONCLUSION: Chromosomal status analysis based on TE biopsy does not accurately reflect the chromosomal status of the whole blastocyst. The chromosomal status is usually the same between the TE and remaining blastocyst cells in cases diagnosed as euploid or aneuploid with no mosaicism. However, mosaic blastocysts and those with other types of structural rearrangements have a higher risk of inconsistency, warranting caution during embryo selection.

Wide application of a novel topoisomerase I inhibitor-based drug conjugation technology.

To establish a novel and widely applicable payload-linker technology for antibody-drug conjugates (ADCs), we have focused our research on applying exatecan mesylate (DX-8951f), a potent topoisomerase I inhibitor, which exhibits extensive antitumor activity as well as significant myelotoxicity, as the payload part. Through this study, we discovered a promising exatecan derivative (DX-8951 derivative, DXd), that has the characteristics of low membrane permeability and shows considerably less myelotoxicity than that shown by exatecan mesylate in an in vitro human colony forming unit-granulocyte macrophage assay. DXd was further used for drug conjugation by using commercially or clinically useful monoclonal antibodies to evaluate the potency of the ADC. The result revealed that the DXd-ADCs targeting CD30, CD33, and CD70 were effective against each of their respective target-expressing tumor cell lines. Moreover, a novel DXd-ADC targeting B7-H3, which is a new target for ADCs, also showed potent antitumor efficacy both in vitro and in vivo. In conclusion, this study showed that this novel topoisomerase I inhibitor-based ADC technology is widely applicable to a diverse number of antibodies and is expected to mitigate myelotoxicity, thereby possibly resulting in better safety profiles than that of existing ADC technologies.

Shape of the first mitotic spindles impacts multinucleation in human embryos.

During human embryonic development, early cleavage-stage embryos are more susceptible to errors. Studies have shown that many problems occur during the first mitosis, such as direct cleavage, chromosome segregation errors, and multinucleation. However, the mechanisms whereby these errors occur during the first mitosis in human embryos remain unknown. To clarify this aspect, in the present study, we image discarded living human two-pronuclear stage zygotes using fluorescent labeling and confocal microscopy without microinjection of DNA or mRNA and investigate the association between spindle shape and nuclear abnormality during the first mitosis. We observe that the first mitotic spindles vary, and low-aspect-ratio-shaped spindles tend to lead to the formation of multiple nuclei at the 2-cell stage. Moreover, we observe defocusing poles in many of the first mitotic spindles, which are strongly associated with multinucleation. Additionally, we show that differences in the positions of the centrosomes cause spindle abnormality in the first mitosis. Furthermore, many multinuclei are modified to form mononuclei after the second mitosis because the occurrence of pole defocusing is firmly reduced. Our study will contribute markedly to research on the occurrence of mitotic errors during the early cleavage of human embryos.

Live visualisation of electrolytes during mouse embryonic development using electrolyte indicators.

Studies have shown that some electrolytes, including Na+ and K+, play important roles in embryonic development. However, these studies evaluated these electrolytes by using inhibitors or knockout mice, with no mention on the changes in the intracellular electrolyte concentrations during embryogenesis. In this study, we used the electrolyte indicators CoroNa Green AM and ION Potassium Green-2 AM to directly visualise intracellular concentrations of Na+ and K+, respectively, at each embryonic developmental stage in mouse embryos. We directly observed intracellular electrolyte concentrations at the morula, blastocyst, and hatching stages. Our results revealed dynamic changes in intracellular electrolyte concentrations; we found that the intracellular Na+ concentration decreased, while K+ concentration increased during blastocoel formation. The degree of change in intensity in response to ouabain, an inhibitor of Na+/K+ ATPase, was considered to correspond to the degree of Na+/K+ ATPase activity at each developmental stage. Additionally, after the blastocyst stage, trophectoderm cells in direct contact with the blastocoel showed higher K+ concentrations than in direct contact with inner cell mass, indicating that Na+/K+ ATPase activity differs depending on the location in the trophectoderm. This is the first study to use CoroNa Green AM and ION Potassium Green-2 AM in mouse embryos and visualise electrolytes during embryonic development. The changes in electrolyte concentration observed in this study were consistent with the activity of Na+/K+ ATPase reported previously, and it was possible to image more detailed electrolyte behaviour in embryo cells. This method can be used to improve the understanding of cell physiology and is useful for future embryonic development studies.

Cell-free DNA in spent culture medium effectively reflects the chromosomal status of embryos following culturing beyond implantation compared to trophectoderm biopsy.

This prospective study evaluated the accuracy of non-invasive preimplantation genetic testing for aneuploidy (niPGT-A) using cell-free DNA in spent culture medium, as well as that of preimplantation genetic testing for aneuploidy (PGT-A) using trophectoderm (TE) biopsy after culturing beyond implantation. Twenty frozen blastocysts donated by 12 patients who underwent IVF at our institution were investigated. Of these, 10 were frozen on day 5 and 10 on day 6. Spent culture medium and TE cells were collected from each blastocyst after thawing, and the embryos were cultured in vitro for up to 10 days. The outgrowths after culturing beyond implantation were sampled and subjected to chromosome analysis using next-generation sequencing. Chromosomal concordance rate, sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), false-positive rate (FPR), and false-negative rate (FNR) of niPGT-A and PGT-A against each outgrowth were analyzed. The concordance rate between the niPGT-A and outgrowth samples was 9/16 (56.3%), and the concordance rate between the PGT-A and outgrowth samples was 7/16 (43.8%). NiPGT-A exhibited 100% sensitivity, 87.5% specificity, 88.9% PPV, 100% NPV, 12.5% FPR, and 0% FNR. PGT-A exhibited 87.5% sensitivity, 77.8% specificity, 87.5% PPV, 75% NPV, 14.3% FPR, and 22.2% FNR. NiPGT-A may be more accurate than PGT-A in terms of ploidy diagnostic accuracy in outgrowths.

Kinetics of meiotic maturation in oocytes from unstimulated ovaries and duration of pronucleus presence and preimplantation development.

OBJECTIVE: To evaluate the meaning of meiotic maturation kinetics and duration of pronucleus presence (DPP) for parthenogenetic activation outcome. DESIGN: Retrospective study. SETTING: University hospital. PATIENT(S): Eight patients with endometrioid adenocarcinoma and 65 patients who underwent in vitro fertilization (IVF) with intracytoplasmic sperm injection (ICSI). INTERVENTION(S): After collection of oocytes from nonstimulated ovaries of patients with endometrioid adenocarcinoma, in vitro maturation (IVM) and parthenogenetic activation performed with time-lapse imaging; after ICSI, embryos similarly incubated with time-lapse imaging. MAIN OUTCOME MEASURE(S): Timing of the release of the first polar body (fPB), DPP, and developmental stage with IVM and parthenogenetic activation; after ICSI, assessment of DPP and preimplantation developmental stage. RESULT(S): With IVM, 55.2% of oocytes matured; 53.1% of fPBs were released within 24 hours, and 46.9% of fPBs were released after 24 hours. Regarding developmental stage, oocytes that released fPB later during IVM tended to develop more than oocytes that released the fPB within 24 hours. For embryos from parthenogenetic activation the DPP was statistically significantly shorter than the DPP of embryos from ICSI. With ICSI, the DPP was statistically significantly shorter in embryos that developed to ≥8 cells than embryos whose final development included ≤7 cells. The development rate in parthenogenetic activation was statistically significantly lower than that in ICSI. CONCLUSION(S): Embryo development is negatively affected by DPP that is too short or too long. When the DPP was short with parthenogenetic activation, embryo development did not proceed, indicating that DPP is an important determinant of parthenogenetic activation outcomes as with the timing of fPB release.

Human frozen-thawed blastocyst morphokinetics observed using time-lapse cinematography reflects the number of trophectoderm cells.

Recent studies reported morphokinetic indices for optimal selection of embryos in assisted reproductive technology (ART). The morphokinetics in blastocyst stage include the collapse and re-expansion rates after thawing. However, evaluation methods using these morphokinetics have not been established, mainly because the underlying molecular mechanisms remain unclarified. In this study, we focused on the relationship between these morphokinetic observation of the blastocyst behaviour and the number of cells constituting the blastocyst. We evaluated 38 surplus human frozen-thawed blastocysts using time-lapse cinematography and recorded their expansion, contraction, and hatching. A total of 28 blastocysts expanded in culture (cross-sectional area ≥ 5,000 π µm2). In comparison to the ones that did not, the expanded group presented significantly more number of inner cell mass (ICM) and trophectoderm (TE) cells, which eventually develop into the fetus and placenta, respectively (ICM: Expanded 10.2 ± 6.3 vs. Non-Expanded 6.0 ± 12.3, p < 0.05; TE: Expanded 165.7 ± 74.8 vs. Non-Expanded 57.0 ± 29.4, p < 0.05). Moreover, a positive correlation was found between the expansion rate (up to 4 h) and the number of TE cells (r = 0.558, p = 0.0021). Additionally, blastocysts that hatched had a significantly higher number of TE cells than those that did not (hatching 225.2 ± 61.2 vs. no hatching 121.1 ± 48.6, p < 0.0001). The number of TE cells per unit of cross-sectional area correlated negatively with the contraction time (r = -0.601, p = 0.0007). No correlation between the number of ICM cells and these morphokinetics was detected. In conclusion, our study demonstrates that different morphokinetics of frozen-thawed blastocysts reflect the number of TE cells. The differentiation of blastocysts containing sufficient TE cells would be beneficial for implantation and prognosis of a subsequent pregnancy. Thus, evaluation of these morphokinetics can be an effective method to screen good embryos for ART.

Evaluation of drug-induced hematotoxicity using novel in vitro monkey CFU-GM and BFU-E colony assays.

In order to evaluate drug-induced hematotoxicity in monkey cells in vitro, colony-forming unit-granulocyte, macrophage (CFU-GM), and burst-forming unit-erythroid (BFU-E) colony assays were established using mononuclear cells in the bone marrow collected from male cynomolgus monkeys. Furthermore, the effects of doxorubicin, chloramphenicol, and linezolid on CFU-GM and BFU-E colony formation were investigated using established monkey CFU-GM and BFU-E colony assays in comparison with those on human CFU-GM and BFU-E colonies acquired from human umbilical cord blood cells. Bone marrow mononuclear cells were collected from the ischial or iliac bone of male cynomolgus monkeys. The cells were subsequently processed by density gradient separation at 1.067, 1.070, or 1.077 g/mL for CFU-GM or 1.077 g/mL for BFU-E, and then cultured in methylcellulose medium for 9 or 13 days, respectively. A sufficient number of CFU-GM colonies were formed from mononuclear cells processed at a density of 1.070 g/mL. Moreover, the number of BFU-E colonies from the cells processed at a density of 1.077 g/mL was sufficient for the colony assay. The number of CFU-GM or BFU-E colonies decreased after treatment with the drugs of interest in a concentration-dependent manner. Compared with human CFU-GM, monkey CFU-GM were more sensitive to chloramphenicol and resistant to doxorubicin, whereas monkey BFU-E were more sensitive to all compounds in comparison to the sensitivity of human BFU-E. In conclusion, monkey CFU-GM and BFU-E colony assays were established and considered useful tools to evaluate the differences in drug-induced hematotoxicity between species.

The location of "8"-shaped hatching influences inner cell mass formation in mouse blastocysts.

The hatching of a blastocyst where the blastocyst portions on the inside and the outside of the zona pellucida feature a figure-of-eight shape is termed "8"-shaped hatching; this type of hatching has been reported to affect the proper presentation of the inner cell mass (ICM) in both human and mouse embryos. Here, our aim was to investigate the factors that affect ICM presentation during "8"-shaped hatching. We performed IVF by using B6D2F1 female mice and ICR male mice, and used the 104 captured blastocysts. Embryos were maintained in KSOM at 37°C in a 5% CO2, 5% O2, and 90% N2 environment, and their growth behavior was monitored individually and continuously using time-lapse cinematography. At 120 h after insemination, embryos were immunostained and examined under a confocal microscope. We used the hatching form to identify "8"-shaped hatching, and we classified the "8"-shaped-hatching blastocysts into two groups, one in which the hatching site was near the ICM center, and the other in which the hatching site was far from the ICM center. We measured each group for ICM size and the number of Oct3/4-positive cells. Of the 95 hatching or hatched embryos, 74 were "8"-shaped-hatching blastocysts, and in these embryos, the ICM was significantly wider when the hatching site was near the ICM than when the hatching site was far from the ICM (P = 0.0091). Moreover, in the "8"-shaped-hatching blastocysts in which the ICM was included in the blastocyst portion outside the zona pellucida-the portion defined as the "outside blastocyst"-after the collapse of this outside blastocyst, the ICM adhered to the trophectoderm of the outside blastocyst, opposite the hatching site. Our results indicate that in "8"-shaped-hatching blastocysts, the hatching site and the collapse of outside blastocyst affect ICM formation. Thus, the assessment of "8"-shaped hatching behaviors could yield indices for accurately evaluating embryo quality.

Tienilic acid enhances hyperbilirubinemia in Eisai hyperbilirubinuria rats through hepatic multidrug resistance-associated protein 3 and heme oxygenase-1 induction.

We demonstrated that tienilic acid, a diuretic drug withdrawn from the market because of hepatic failure, enhanced hyperbilirubinemia in Eisai hyperbilirubinuria rats (EHBR) with a defect of canalicular multidrug resistance-associated protein 2 (Mrp2). In contrast, no remarkable changes were noted in Sprague-Dawley (SD) rats, the parent strain for EHBR. To investigate a mechanism underlying this enhanced hyperbilirubinemia, we focused on comprehensive effects of tienilic acid on clinicopathological aspects and expression of hepatic transporters. Other than eventual hyperbilirubinemia with slightly increased biliary bilirubin, a single oral treatment of EHBR with tienilic acid at 300 mg/kg caused no changes in serum alanine aminotransferase and alkaline phosphatase, bile flow rate and biliary bile acid secretion, or hepatic morphology. In analyses of mRNA expression of the hepatic transporters, elevated Mrp3 expression in EHBR correlated with an increase in serum total bilirubin, suggesting increased bilirubin transport from the liver into the peripheral blood flow. Hepatic heme oxygenase-1 (Ho-1) mRNA, a stress-induced isoform of the rate-limiting enzyme in the catabolism of heme to bilirubin, was markedly upregulated in EHBR at the same dose at which increased serum bilirubin was seen. A time-course study revealed that marked induction of Ho-1 occurred earlier than that of Mrp3, followed by an increase in serum bilirubin. These results suggest that hepatic Mrp3 and Ho-1 may contribute to tienilic acid-enhanced hyperbilirubinemia in EHBR by inducing increased bilirubin transport from the liver into the blood stream, preceded by potentiation of bilirubin formation in the liver.

Effect of body-weight loading onto the articular cartilage on the occurrence of quinolone-induced chondrotoxicity in juvenile rats.

The effect of body-weight loading onto the articular cartilage on the occurrence of chondrotoxicity was investigated in male juvenile Sprague-Dawley rats given ofloxacin (OFLX) orally once at 900 mg/kg. Just after dosing of OFLX, hindlimb unloading was performed for 0, 2, 4, or 8 h by a tail-suspension method. Animals were sacrificed at 8h post-dose, and then the distal femoral articular cartilage was subjected to a histological examination and an investigation for gene expression of tumor necrosis factor receptor superfamily, member 12a (Tnfrsf12a); prostaglandin-endoperoxide synthase 2 (Ptgs2); plasminogen activator, urokinase receptor (Plaur); and matrix metalloproteinase 3 (Mmp3) by qRT-PCR analysis. As a result, cartilage lesions and up-regulations of these 4 genes that were seen in rats without the tail suspension were not observed in rats with the 8-h tail suspension, and a tendency to decrease in the incidence of the cartilage lesions and the gene expression was noted in a tail-suspension time dependent manner. Our results clearly indicate that body-weight loading onto the cartilage is necessary to induce cartilage lesions and gene expression of Tnfrsf12a, Ptgs2, Plaur, and Mmp3 in juvenile rats treated with OFLX.