RESUMO
STUDY QUESTION: What have we learnt after 10 years of electronic witnessing? SUMMARY ANSWER: When applied correctly, an electronic witnessing system can replace manual witnessing in the medically assisted reproduction lab to prevent sample mix-up. WHAT IS KNOWN ALREADY: Electronic witnessing systems have been implemented to improve the correct identification, processing, and traceability of biological materials. When non-matching samples are simultaneously present in a single workstation, a mismatch event is generated to prevent sample mix-up. STUDY DESIGN, SIZE, DURATION: This evaluation investigates the mismatch and administrator assign rate over a 10-year period (March 2011-December 2021) with the use of an electronic witnessing system. Radiofrequency identification tags and barcodes were used for patient and sample identification. Since 2011, IVF and ICSI cycles and frozen embryo transfer cycles (FET) were included; IUIs cycles were included since 2013. PARTICIPANTS/MATERIALS, SETTING, METHODS: The total number of tags and witnessing points were recorded. Witnessing points in a particular electronic witnessing system represent all the actions that have been performed from gamete collection through embryo production, to cryopreservation and transfer. Mismatches and administrator assigns were collected and stratified per procedure (sperm preparation, oocyte retrieval, IVF/ICSI, cleavage stage embryo or blastocyst embryo biopsy, vitrification and warming, embryo transfer, medium changeover, and IUI). Critical mismatches (such as mislabelling or non-matching samples within one work area) and critical administrator assigns (such as samples not identified by the electronic witnessing system and unconfirmed witnessing points) were selected. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 109â655 cycles were included: 53â023 IVF/ICSI, 36â347 FET, and 20â285 IUI cycles. The 724â096 used tags, led to a total of 849â650 witnessing points. The overall mismatch rate was 0.251% (2132/849â650) per witnessing point and 1.944% per cycle. In total, 144 critical mismatches occurred over the different procedures. The yearly mean critical mismatch rate was 0.017 ± 0.007% per witnessing point and 0.129 ± 0.052% per cycle. The overall administrator assign rate was 0.111% (940/849â650) per witnessing point and 0.857% per cycle, including 320 critical administrator assigns. The yearly mean critical administrator assign rate was 0.039 ± 0.010% per witnessing point and 0.301 ± 0.069% per cycle. Overall mismatch and administrator assign rates remained fairly stable during the evaluated time period. Sperm preparation and IVF/ICSI were the procedures most prone to critical mismatch and administrator assigns. LIMITATIONS, REASONS FOR CAUTION: The procedures and methods of integration of an electronic witnessing system may vary from one laboratory to another and result in differences in the potential risks related to sample identification. Individual embryos cannot (yet) be identified by such a system; this makes extra manual witnessing indispensable at certain critical steps where potential errors are not recorded. The electronic witnessing system still needs to be used in combination with manual labelling of both the bottom and lid of dishes and tubes to guarantee correct assignment in case of malfunction or incorrect use of radiofrequency identification tags. WIDER IMPLICATIONS OF THE FINDINGS: Electronic witnessing is considered to be the ultimate tool to safeguard correct identification of gametes and embryos. But this is only possible when used correctly, and proper training and attention of the staff is required. It may also induce new risks, i.e. blind witnessing of samples by the operator. STUDY FUNDING/COMPETING INTEREST(S): No funding was either sought or obtained for this study. J.S. presents webinars on RIW for CooperSurgical. The remaining authors have nothing to declare. TRIAL REGISTRATION NUMBER: N/A.
Assuntos
Técnicas de Reprodução Assistida , Sêmen , Gravidez , Feminino , Masculino , Humanos , Taxa de Gravidez , Transferência Embrionária/métodos , Reprodução , Estudos Retrospectivos , Fertilização in vitro/métodosRESUMO
STUDY QUESTION: Which processes and transcription factors specify the first and second lineage segregation events during human preimplantation development? SUMMARY ANSWER: Differentiation into trophectoderm (TE) cells can be initiated independently of polarity; moreover, TEAD1 and YAP1 co-localize in (precursor) TE and primitive endoderm (PrE) cells, suggesting a role in both the first and the second lineage segregation events. WHAT IS KNOWN ALREADY: We know that polarity, YAP1/GATA3 signalling and phospholipase C signalling play a key role in TE initiation in compacted human embryos, however, little is known about the TEAD family of transcription factors that become activated by YAP1 and, especially, whether they play a role during epiblast (EPI) and PrE formation. In mouse embryos, polarized outer cells show nuclear TEAD4/YAP1 activity that upregulates Cdx2 and Gata3 expression while inner cells exclude YAP1 which upregulates Sox2 expression. The second lineage segregation event in mouse embryos is orchestrated by FGF4/FGFR2 signalling which could not be confirmed in human embryos; TEAD1/YAP1 signalling also plays a role during the establishment of mouse EPI cells. STUDY DESIGN, SIZE, DURATION: Based on morphology, we set up a development timeline of 188 human preimplantation embryos between Day 4 and 6 post-fertilization (dpf). The compaction process was divided into three subgroups: embryos at the start (C0), during (C1), and at the end (C2) of, compaction. Inner cells were identified as cells that were entirely separated from the perivitelline space and enclosed by cellular contacts on all sides. The blastulation process was divided into six subgroups, starting with early blastocysts with sickle-cell shaped outer cells (B0) and further on, blastocysts with a cavity (B1). Full blastocysts (B2) showed a visible ICM and outer cells referred to as TE. Further expanded blastocysts (B3) had accumulated fluid and started to expand due to TE cell proliferation and zona pellucida (ZP) thinning. The blastocysts then significantly expanded further (B4) and started to hatch out of the ZP (B5) until they were fully hatched (B6). PARTICIPANTS/MATERIALS, SETTING, METHODS: After informed consent and the expiration of the 5-year cryopreservation duration, 188 vitrified high quality eight-cell stage human embryos (3 dpf) were warmed and cultured until the required stages were reached. We also cultured 14 embryos that were created for research until the four- and eight-cell stage. The embryos were scored according to their developmental stage (C0-B6) displaying morphological key differences, rather than defining them according to their chronological age. They were fixed and immunostained for different combinations of cytoskeleton (F-actin), polarization (p-ERM), TE (GATA3), EPI (NANOG), PrE (GATA4 and SOX17), and members of the Hippo signalling pathway (YAP1, TEAD1 and TEAD4). We choose these markers based on previous observations in mouse embryos and single cell RNA-sequencing data of human embryos. After confocal imaging (LSM800, Zeiss), we analysed cell numbers within each lineage, different co-localization patterns and nuclear enrichment. MAIN RESULTS AND THE ROLE OF CHANCE: We found that in human preimplantation embryos compaction is a heterogeneous process that takes place between the eight-cell to the 16-cell stages. Inner and outer cells are established at the end of the compaction process (C2) when the embryos contain up to six inner cells. Full apical p-ERM polarity is present in all outer cells of compacted C2 embryos. Co-localization of p-ERM and F-actin increases steadily from 42.2% to 100% of the outer cells, between C2 and B1 stages, while p-ERM polarizes before F-actin (P < 0.00001). Next, we sought to determine which factors specify the first lineage segregation event. We found that 19.5% of the nuclei stain positive for YAP1 at the start of compaction (C0) which increases to 56.1% during compaction (C1). At the C2 stage, 84.6% of polarized outer cells display high levels of nuclear YAP1 while it is absent in 75% of non-polarized inner cells. In general, throughout the B0-B3 blastocyst stages, polarized outer/TE cells are mainly positive for YAP1 and non-polarized inner/ICM cells are negative for YAP1. From the C1 stage onwards, before polarity is established, the TE marker GATA3 is detectable in YAP1 positive cells (11.6%), indicating that differentiation into TE cells can be initiated independently of polarity. Co-localization of YAP1 and GATA3 increases steadily in outer/TE cells (21.8% in C2 up to 97.3% in B3). Transcription factor TEAD4 is ubiquitously present throughout preimplantation development from the compacted stage onwards (C2-B6). TEAD1 displays a distinct pattern that coincides with YAP1/GATA3 co-localization in the outer cells. Most outer/TE cells throughout the B0-B3 blastocyst stages are positive for TEAD1 and YAP1. However, TEAD1 proteins are also detected in most nuclei of the inner/ICM cells of the blastocysts from cavitation onwards, but at visibly lower levels as compared to that in TE cells. In the ICM of B3 blastocysts, we found one main population of cells with NANOG+/SOX17-/GATA4- nuclei (89.1%), but exceptionally we found NANOG+/SOX17+/GATA4+ cells (0.8%). In seven out of nine B3 blastocysts, nuclear NANOG was found in all the ICM cells, supporting the previously reported hypothesis that PrE cells arise from EPI cells. Finally, to determine which factors specify the second lineage segregation event, we co-stained for TEAD1, YAP1, and GATA4. We identified two main ICM cell populations in B4-6 blastocysts: the EPI (negative for the three markers, 46.5%) and the PrE (positive for the three markers, 28.1%) cells. We conclude that TEAD1 and YAP1 co-localise in (precursor) TE and PrE cells, indicating that TEAD1/YAP1 signalling plays a role in the first and the second lineage segregation events. LIMITATIONS, REASONS FOR CAUTION: In this descriptive study, we did not perform functional studies to investigate the role of TEAD1/YAP1 signalling during the first and second lineage segregation events. WIDER IMPLICATIONS OF THE FINDINGS: Our detailed roadmap on polarization, compaction, position and lineage segregation events during human preimplantation development paves the way for further functional studies. Understanding the gene regulatory networks and signalling pathways involved in early embryogenesis could ultimately provide insights into why embryonic development is sometimes impaired and facilitate the establishment of guidelines for good practice in the IVF lab. STUDY FUNDING/COMPETING INTERESTS: This work was financially supported by Wetenschappelijk Fonds Willy Gepts (WFWG) of the University Hospital UZ Brussel (WFWG142) and the Fonds Wetenschappelijk Onderzoek-Vlaanderen (FWO, G034514N). M.R. is doctoral fellow at the FWO. The authors have no conflicts of interest to declare. TRIAL REGISTRATION NUMBER: N/A.
Assuntos
Actinas , Blastocisto , Gravidez , Feminino , Humanos , Camundongos , Animais , Actinas/metabolismo , Blastocisto/metabolismo , Desenvolvimento Embrionário/fisiologia , Fatores de Transcrição/genética , Embrião de Mamíferos/metabolismo , Fatores de Transcrição de Domínio TEARESUMO
STUDY QUESTION: Are human trophectoderm (TE) cells committed or still able to develop into inner cell mass (ICM) cells? SUMMARY ANSWER: Human full blastocyst TE cells still have the capacity to develop into ICM cells expressing the pluripotency marker NANOG, thus they are not yet committed. WHAT IS KNOWN ALREADY: Human Day 5 full blastocyst TE cells express the pluripotency markers POU5F1, SOX2 and SALL4 as well as the TE markers HLA-G and KRT18 but not yet CDX2, therefore their developmental direction may not yet be definite. STUDY DESIGN, SIZE, DURATION: The potency of human blastocyst TE cells was investigated by determining their in vitro capacity to develop into a blastocyst with ICM cells expressing NANOG; TE cells were isolated either by aspiration under visual control or after labeling with fluorescent 594-wheat germ agglutinin. Further on, aspirated TE cells were also labeled with fluorescent PKH67 and repositioned in the center of the original embryo. PARTICIPANTS/MATERIALS, SETTING, METHODS: Human preimplantation embryos were used for research after obtaining informed consent from IVF patients. The experiments were approved by the Local Ethical Committee and the 'Belgian Federal Committee on medical and scientific research on embryos in vitro'. Outer cells were isolated and reaggregated by micromanipulation. Reconstituted embryos were analyzed by immunocytochemistry. MAIN RESULTS AND THE ROLE OF CHANCE: Isolated and reaggregated TE cells from full human blastocysts are able to develop into blastocysts with ICM cells expressing the pluripotency marker NANOG. Moreover, the majority of the isolated TE cells which were repositioned in the center of the embryo do not sort back to their original position but integrate within the ICM and start to express NANOG. LIMITATIONS, REASONS FOR CAUTION: Owing to legal and ethical restrictions, manipulated human embryos cannot be transferred into the uterus to determine their totipotent capacity. The definitive demonstration that embryos reconstructed with TE cells are a source of pluripotent cells is to obtain human embryonic stem cell 'like' line(s), which will allow full characterization of the cells. WIDER IMPLICATIONS OF THE FINDINGS: Our finding has important implications in reproductive medicine and stem cell biology because TE cells have a greater developmental potential than assumed previously. STUDY FUNDING/COMPETING INTEREST(S): Scientific Research Foundation-Flanders (FWO-Vlaanderen) and Research Council (OZR) of the Vrije Universiteit Brussel. None of the authors declared a conflict of interest.
Assuntos
Blastocisto/citologia , Ectogênese , Células-Tronco Embrionárias/citologia , Células-Tronco Pluripotentes/citologia , Blastocisto/metabolismo , Massa Celular Interna do Blastocisto/citologia , Massa Celular Interna do Blastocisto/metabolismo , Separação Celular , Técnicas de Cultura Embrionária , Células-Tronco Embrionárias/metabolismo , Corantes Fluorescentes/química , Proteínas de Homeodomínio/metabolismo , Humanos , Imuno-Histoquímica , Micromanipulação , Proteína Homeobox Nanog , Células-Tronco Pluripotentes/metabolismo , Antígenos Embrionários Estágio-Específicos/metabolismo , Trofoblastos/citologia , Trofoblastos/metabolismoRESUMO
In this study, we deep-sequenced the mtDNA of human embryonic and induced pluripotent stem cells (hESCs and hiPSCs) and their source cells and found that the majority of variants pre-existed in the cells used to establish the lines. Early-passage hESCs carried few and low-load heteroplasmic variants, similar to those identified in oocytes and inner cell masses. The number and heteroplasmic loads of these variants increased with prolonged cell culture. The study of 120 individual cells of early- and late-passage hESCs revealed a significant diversity in mtDNA heteroplasmic variants at the single-cell level and that the variants that increase during time in culture are always passenger to the appearance of chromosomal abnormalities. We found that early-passage hiPSCs carry much higher loads of mtDNA variants than hESCs, which single-fibroblast sequencing proved pre-existed in the source cells. Finally, we show that these variants are stably transmitted during short-term differentiation.