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Live cell labeling of various organelles is of great demand in the research field of cell biology. However, current approaches often lack an optimal balance between efficiency and versatility. We took advantage of chemical modified mRNA to express various organelle located fluorescent proteins. This approach facilitated highly efficient multi-organelle labeling across diverse cell types, both in vitro and in vivo. The application of mRNA-based organelle markers offers faster and more efficient transfection and expression compared to plasmids. When expressing fluorescent proteins in neuronal cells, it is faster and safer compared to viral vectors. Notably, this approach excels in rapidly labeling organelles, making it highly effective for dynamic live cell imaging applications. By leveraging this tool, we unveiled the unique behaviors of mitochondria and the endoplasmic reticulum during spike protein-mediated cell fusion, a critical event in SARS-CoV-2 viral entry. Thus, our results demonstrate the potency of mRNA-encoded fluorescent proteins as powerful tools for advancing biological research., (© 2024 Federation of American Societies for Experimental Biology.)

Research Question: Embryo blastomeres and the zona pellucida are occasionally damaged during vitrification; is this a result of crack-induced mechanical damage in the glass state, caused by external bending of the device?, Design: A stereomicroscope was used to observe external bending-induced cracks in a cryoprotectant. Thereafter, 309 human cleavage-stage embryos derived from abnormally fertilized eggs were used to assess embryo damage under two external bending conditions: forward bending and backward bending, with three bending degrees applied. Three distinct embryo positions were used to examine the correlation between bending and embryo damage. Damage was assessed by looking at blastomere lysis rates, and overall rates of damaged and surviving embryos., Results: A series of parallel cracks were identified in the cryoprotectant used for external bending, which led to damage to the embryo blastomeres. Compared with forward bending and control, the embryos were found to be more easily damaged by backward bending, indicated by significantly higher blastomere lysis and embryo damage rates, and lower embryo survival rate of backward bending than forward bending (P < 0.001). The degree of embryo damage also increased as the degree of external forces increased. Embryo position correlated with degree of embryo damage., Conclusions: Cryoprotectant crack-induced damage was identified as the cause of embryo damage. Mechanical damage to the glass state occurs because of improper external bending of the cryodevice strip in liquid nitrogen during vitrification. To prevent damage, bending of the strip should be avoided and the embryos should be placed near the tip of the strip., (Copyright © 2023 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)

Mutations in mitochondrial DNA (mtDNA) contribute to a variety of serious multi-organ human diseases, which are strictly inherited from the maternal germline. However, there is currently no curative treatment. Attention has been focused on preventing the transmission of mitochondrial diseases through mitochondrial replacement (MR) therapy, but levels of mutant mtDNA can often unexpectedly undergo significant changes known as mitochondrial genetic drift. Here, we proposed a novel strategy to perform spindle-chromosomal complex transfer (SCCT) with maximal residue removal (MRR) in metaphase II (MII) oocytes, thus hopefully eliminated the transmission of mtDNA diseases. With the MRR procedure, we initially investigated the proportions of mtDNA copy numbers in isolated karyoplasts to those of individual oocytes. Spindle-chromosomal morphology and copy number variation (CNV) analysis also confirmed the safety of this method. Then, we reconstructed oocytes by MRR-SCCT, which well developed to blastocysts with minimal mtDNA residue and normal chromosomal copy numbers. Meanwhile, we optimized the manipulation order between intracytoplasmic sperm injection (ICSI) and SCC transfer and concluded that ICSI-then-transfer was conducive to avoid premature activation of reconstructed oocytes in favor of normal fertilization. Offspring of mice generated by embryos transplantation in vivo and embryonic stem cells derivation further presented evidences for competitive development competence and stable mtDNA carryover without genetic drift. Importantly, we also successfully accomplished SCCT in human MII oocytes resulting in tiny mtDNA residue and excellent embryo development through MRR manipulation. Taken together, our preclinical mouse and human models of the MRR-SCCT strategy not only demonstrated efficient residue removal but also high compatibility with normal embryo development, thus could potentially be served as a feasible clinical treatment to prevent the transmission of inherited mtDNA diseases., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Liao et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The second polar body (PB2) transfer in assisted reproductive technology is regarded as the most promising mitochondrial replacement scheme for preventing the mitochondrial disease inheritance owing to its less mitochondrial carryover and stronger operability. However, the mitochondrial carryover was still detectable in the reconstructed oocyte in conventional second polar body transfer scheme. Moreover, the delayed operating time would increase the second polar body DNA damage. In this study, we established a spindle-protrusion-retained second polar body separation technique, which allowed us to perform earlier second polar body transfer to avoid DNA damage accumulation. We could also locate the fusion site after the transfer through the spindle protrusion. Then, we further eliminated the mitochondrial carryover in the reconstructed oocytes through a physically based residue removal method. The results showed that our scheme could produce a nearly normal proportion of normal-karyotype blastocysts with further reduced mitochondrial carryover, both in mice and humans. Additionally, we also obtained mouse embryonic stem cells and healthy live-born mice with almost undetectable mitochondrial carryover. These findings indicate that our improvement in the second polar body transfer is conducive to the development and further mitochondria carryover elimination of reconstructed embryos, which provides a valuable choice for future clinical applications of mitochondrial replacement., Competing Interests: The authors declare no conflicts of interest., (© 2023 The Authors. MedComm published by Sichuan International Medical Exchange & Promotion Association (SCIMEA) and John Wiley & Sons Australia, Ltd.)

Background: 0PN zygotes have a low cleavage rate, and the clinical outcomes of cleavage-stage embryo transfers are unsatisfactory. Blastocyst culturing is used to screen 0PN embryos, but whether the cell number of 0PN embryos on day 3 affects the clinical outcomes following single blastocyst transfer is unknown and would be helpful in evaluating the clinical value of these embryos., Methods: This retrospective study compared 46,804 0PN zygotes, 242 0PN frozen-thawed single blastocyst transfers, and 92 corresponding 0PN singletons with 232,441 2PN zygotes, 3563 2PN frozen-thawed single blastocyst transfers, and 1250 2PN singletons from January 2015 to October 2019 at a tertiary-care academic medical centre. The 0PN and 2PN embryos were divided into two groups: the group with < 6 cells on day 3 and that with ≥ 6 cells. Embryo development, subsequent pregnancy and neonatal outcomes were compared between the two groups., Results: The cleavage and available blastocyst rates of the 0PN zygotes were much lower than those of the 2PN zygotes (25.9% vs. 97.4%, P < 0.001; 13.9% vs. 23.4%, P < 0.001). In the < 6 cells group, the available blastocyst rate of the cleaved 0PN embryos was significantly lower than that of the 2PN embryos (2.5% vs. 12.7%, P < 0.001). However, in the ≥ 6 cells group, the available blastocyst rate of the 0PN cleaved embryos significantly improved, although it was slightly lower than that of the 2PN embryos (33.9% vs. 35.7%, P = 0.014). Importantly, compared to those of the 2PN single blastocyst transfers, the clinical pregnancy rate, live birth rate, Z-score and malformation rate of the 0PN single blastocyst transfers were not significantly different in either the < 6 cells group (30.4% vs. 39.8%, P = 0.362; 30.4% vs. 31.3%, P = 0.932; 0.89 ± 0.90 vs. 0.42 ± 1.02, P = 0.161; 0% vs. 2.6%, P = 1.000) or the ≥ 6 cells group (50.7% vs. 46.6%, P = 0.246; 39.7% vs. 38.3%, P = 0.677; 0.50 ± 1.23 vs. 0.47 ± 1.11, P = 0.861; 2.4% vs. 1.8%, P = 1.000)., Conclusions: The cell number on day 3 of 0PN embryos affected the subsequent formation of blastocysts but did not influence the subsequent pregnancy and neonatal outcomes of 0PN single blastocyst transfers, which may be beneficial to clinicians counselling patients on the clinical value of 0PN embryos., (© 2022. The Author(s).)

Background: It is unclear whether we should focus attention on cleavage-stage embryo quality and embryo development speed when transferring single particular grade vitrified-warmed blastocysts, especially poor-quality blastocysts (grade "C")., Method: This retrospective study considered 3386 single vitrified-warmed blastocyst transfer cycles from January 2010 to December 2017. They were divided into group 1 (AA/AB/BA, n = 374), group 2 (BB, n = 1789), group 3 (BC, n = 901), and group 4 (CB, n = 322). The effects of cleavage-stage embryo quality and embryo development speed were measured in terms of clinical pregnancy and live birth rates in each group., Results: Pregnancy outcomes showed a worsening trend from groups 1 to 4; the proportion of embryos with better cleavage-stage quality and faster development speed decreased. In group 1, only the blastocyst expansion degree 3 was a negative factor in the clinical pregnancy rate (odds ratio (OR) [95% confidence interval (CI)]: 0.233 [0.091-0.595]) and live birth rate (0.280 [0.093-0.884]). In the other groups (BB, BC, and CB), blastocysts frozen on day 5 had significantly better clinical pregnancy outcomes than those frozen on day 6: 1.373 [1.095-1.722] for group 2, 1.523 [1.055-2.197] for group 3, and 3.627 [1.715-7.671] for group 4. The live birth rate was 1.342 [1.060-1.700] for group 2, 1.544 [1.058-2.253] in group 3, and 3.202 [1.509-6.795] in group 4, all P s < 0.05). The degree of blastocoel expansion three for clinical pregnancy rate in group 2 (0.350 [0.135-0.906], P < 0.05) and day 3 blastomere number (>7) for live birth rate in group 4 (2.455 [1.190-5.063], P < 0.05) were two important factors., Conclusion: We should consider choosing BB/BC/CB grade blastocysts frozen on day 5, CB grade blastocysts with day 3 blastomere numbers (>7), and AA/AB/BA grade blastocysts with degrees of expansion (≥4) to obtain better pregnancy outcomes., (Copyright © 2020 Shen, Long, Gao, Guo, Xie, Chen, Cong, Wang, Li, Si, Zhao, Lyu, Kuang and Wang.)

The donor cells from different individuals and with different foreign genes introduced were investigated to determine their effects on the efficiency of somatic cell nuclear transfer (SCNT). The bovine ear fibroblast from different individuals was isolated, cultured, and then transfected with foreign genes to establish the stable cell lines, which were used as donor cells for nuclear transfer. The oocytes were obtained through ovum pick up operation. After in vitro maturation, the M II phase oocytes were selected as receptors for nuclear transfer. The reconstructed embryos were cultured in vitro and observed at 2 h, 48 h, and 7 days after transfer to assess the rate of fusion using cleaved and blastocyst as the parameters of SCNT efficiency. The donor cells from different individuals (04036, 06081, 06088, and 06129) had no obvious effect on the fusion and cleaved rate, whereas there was significant difference in the blastocyst rate (P<0.05), and the rate was 62.3%, 37.0%, 35.1%, and 15.6%, respectively. There was no significant difference among the rate of fusion, cleaved and blastocyst in donor cells with different foreign genes (P>0.05). It was concluded that the genetic background of the donor cells could affect the efficiency of SCNT, while the introduction of foreign genes into the donor cells had no obvious effect on the efficiency. This study provides useful information for the SCNT and would benefit in promoting the efficiency.