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The developmental process of the mammalian blastocyst involves the intricate interplay of cellular and molecular mechanisms, including electrolyte dynamics within the trophectoderm (TE). We hypothesized that sodium (Na + ) is actively transported from the TE into the blastocyst cavity, driving water influx and promoting blastocyst expansion. In this study, we investigated the dynamics of Na + concentration in the TE of mouse embryos using sodium-binding benzofuran isophthalate (SBFI), a dual-wavelength Na + -sensitive fluorescent indicator. Observations revealed three distinct patterns of Na + dynamics, each correlating with variations in blastocyst cross-sectional area and developmental outcomes. Embryos exhibiting an initial decrease followed by stabilization of Na + concentration (Group A) demonstrated the highest rates of hatching, suggesting a relationship between Na + flux and successful embryonic development. In contrast, embryos with transient increases (Group B) displayed reduced hatching rates and developmental progression. Further, the inhibition of Na + /K + -ATPase activity disrupted Na + flux and blastocyst cavity expansion, emphasizing its critical role in blastocyst formation. This study highlights the potential of dual-wavelength imaging for elucidating electrolyte dynamics in preimplantation embryos and its implications for optimizing embryo culture systems in reproductive medicine.

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.

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. Defects in cell division occur often in early embryos, though the mechanisms underlying these errors and their correction remain unclear. Here they develop a method for imaging zygotic chromosomes during human zygotic cleavage and find that the spindle shape influences formation of micronuclei.

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.

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.

Background Uterine adenomyosis is a benign disease, common among women in their 40 and 50 s, characterised by ectopic endometrial tissue in the uterine myometrial layer. Adenomyosis causes infertility and has a negative effect on the outcomes of in vitro fertilisation (IVF)/intracytoplasmic sperm injection (ICSI) embryo transfer (ET) cycles. It has also been reported to have different characteristics depending on the adenomyotic lesion localisation. The effect of its localisation on IVF/ICSI-ET outcomes is unclear. This study aimed to investigate whether adenomyotic lesion localisation, assessed using magnetic resonance imaging (MRI), was associated with outcomes of IVF/ICSI-ET cycles. Methods This multicentre, joint, retrospective cohort study analysed the medical records of 67 infertile patients with adenomyosis who underwent IVF/ICSI with fresh and frozen-thawed ET at five participating facilities from January 2012 to December 2016 and for whom MRI data were available. Fifteen patients were excluded; therefore, the MRI data of 52 patients were evaluated by two radiologists. We assessed the localisation of and classified adenomyotic lesions into advanced (invades the full thickness of the uterine myometrium), extrinsic (localised on the serosal side), and intrinsic (localised on the endometrial side) subtypes. Results There were 40 advanced, nine extrinsic, and three intrinsic cases, and the outcomes of 100, 27, and nine ET cycles, respectively, were analysed. Pregnancy loss/clinical pregnancy and live birth rates of the advanced, extrinsic, and intrinsic groups were 64 % (16/25) and 9 % (9/100), 33.3 % (3/9) and 22.2 % (6/27), and 50 % (1/2) and 11.1 % (1/9), respectively. A logistic regression analysis adjusted for age, prior miscarriage, and body mass index showed that the extrinsic group had fewer pregnancy losses (odds ratio 0.06; 95 % confidence interval [CI]: 0.00–0.54, p = 0.026) and more live births (odds ratio 6.05; 95 % CI: 1.41–29.65, p = 0.018) than the advanced group. Conclusions Adenomyotic lesions exert different effects on IVF/ICSI-ET outcomes. Thus, MRI assessments of adenomyosis in infertile patients are beneficial. Establishment of treatment plans based on adenomyotic lesion localisation should be considered.

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.

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.

The developmental process of the mammalian blastocyst involves the intricate interplay of cellular and molecular mechanisms, including electrolyte dynamics within the trophectoderm (TE). We hypothesized that sodium (Na.sup.+) is actively transported from the TE into the blastocyst cavity, driving water influx and promoting blastocyst expansion. In this study, we investigated the dynamics of Na.sup.+ concentration in the TE of mouse embryos using sodium-binding benzofuran isophthalate (SBFI), a dual-wavelength Na.sup.+ -sensitive fluorescent indicator. Observations revealed three distinct patterns of Na.sup.+ dynamics, each correlating with variations in blastocyst cross-sectional area and developmental outcomes. Embryos exhibiting an initial decrease followed by stabilization of Na.sup.+ concentration (Group A) demonstrated the highest rates of hatching, suggesting a relationship between Na.sup.+ flux and successful embryonic development. In contrast, embryos with transient increases (Group B) displayed reduced hatching rates and developmental progression. Further, the inhibition of Na.sup.+ /K.sup.+ -ATPase activity disrupted Na.sup.+ flux and blastocyst cavity expansion, emphasizing its critical role in blastocyst formation. This study highlights the potential of dual-wavelength imaging for elucidating electrolyte dynamics in preimplantation embryos and its implications for optimizing embryo culture systems in reproductive medicine.