Embryos frozen within a short time of reaching the expanded blastocysts from the early blastocysts have high viability: time-lapse investigation of 5177 blastocysts
Y. KobayashiRie MatsunagaHiromi MoritaRui HasegawaKana IsobeMegumi MiuraM. KamihataShinichi WatanabeTomoko MaedaHiroshi MakinoMasanori OchiToshitaka Horiuchi
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Blastocoel
Live birth
Blastocyst Transfer
Abstract Background Day 5 (D5) blastocyst is generally given priority to transfer than Day 6 (D6) blastocyst, however, which one should be prioritized to transfer when only low-grade D5 and high-grade D6 blastocysts are available? Methods A large retrospective cohort study was carried out to evaluate the live birth rate (LBR) following D5 blastocyst and excellent D6 blastocyst in single frozen-thawed blastocyst transfer (FBT) during Jan 2014 and Dec 2018. The biopsied blastocysts form consecutive PGT-A case series during Feb 2013 to Dec 2021 were performed as a supplementary analysis. Results The LBR was highest in high-grade D5 blastocyst (57.60%) and lowest in low-grade D6 blastocyst (29.72%, vs high-grade D5, aOR 0.38, 95%CI 0.30–0.48, p < 0.001). The LBR achieved in high-grade D6 blastocyst transfer was significantly higher than low-grade D5 blastocyst (50.43% vs 40.70%, aOR 1.54, 95%CI 1.05–2.26, p = 0.027), and is comparable with that in high-grade D5 blastocyst (50.43% vs 57.60%, aOR 0.89, 95%CI 0.61–1.32, p = 0.568). There were no significant differences in preterm birth rate, very preterm birth rate, mean live birth weight, birth weight < 1500g and > 4000g between four cohorts. As for aneuploidy analysis in PGT, there were 54.55% of euploid blastocysts (30/55) among high-grade D6 blastocysts, significantly higher than 41.39% of euploid blastocysts (565/1365) among low-grade D5 blastocyst (p < 0.001). Conclusions Our data suggested Day 6 blastocyst with high morphology grading should be preferred than Day 5 blastocyst with low morphology grading when selecting blastocyst transfer to short the time of conceiving. Trial registration: This study was approved by the hospital institutional ethics committee (No. 2021002).
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Blastocoel
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Human embryonic stem cells (hESC) are derived from the human blastocyst and possess the potential to differentiate into any cell type present in the adult human body. Human ESC are considered to have great potential in regenerative medicine for the future treatment of severe diseases and conditions such as Parkinson’s disease, diabetes, and spinal cord injury. One of today’s challenges in regenerative medicine is to define proper culture conditions for hESC. The natural milieu in the blastocyst may provide clues on how to improve culture conditions, and the aim of the present study was to determine the proteome of the blastocoel fluid and the remaining cells of bovine blastocysts. Bovine blastocysts were produced by in vitro fertilization of oocytes retrieved from slaughterhouse ovaries. The blastocoel from 195 blastocysts (1–8 nL per blastocyst) were isolated by micromanipulation and analysed by nano-HPLC tandem mass spectrometry along with the remaining cells of the blastocyst. Searching the mass spectrometry data against a combined bovine database (SwissProt/TrEMBL), we identified 263 proteins in the blastocoel fluid and 1606 proteins in the cellular compartment of the blastocyst. A Venn diagram showed 124 proteins in overlap between the two compartments of the blastocyst. Several heat shock proteins and specific antioxidants were identified in both the blastocoel and cell material. A selection of proteins identified in the blastocoel fluid is to be tested on hESC in cell culture experiments, with proliferation of undifferentiated cells as the primary endpoint. The results from this study provide new knowledge about early mammalian preimplantation development, and the data can be used in the continued pursue of improving culture conditions for hESC, which further facilitates the clinical application of these cells.
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Abstract Cyclic nucleotide phosphodiesterase (PDE) activity and cAMP amounts were measured in mouse preimplantation embryos at the 1‐cell, 2‐cell, 8‐cell/ morula, and mid‐blastocyst stages. PDE activity remained constant between the 1‐cell and 2‐cell stages. It decreased by the 8‐cell stage and continued to decrease by the mid blastocyst stage to about 14% of the 1‐and 2‐cell values. By contrast, cAMP amounts remained essentially constant at 0.05 fmole/embryo (0.3 μM) from the 1‐cell to the blastocyst stage and increased to 0.175 fmole in the fully expanded blastocyst that was close to hatching. Measurements of embryo volume indicated that intracellular volume remained essentially constant up to the blastocyst stage. The morphological changes in cell shape that accompany differentiation of the trophectoderm and that are coupled with blastocoel expansion decresed the intracellular volume. This decrease resulted in an increase in the cAMP concentration to about 0.4 μM by the mid‐blastocyst stage. Previous studies indicate that either cAMP or TGF‐α/EGF can stimulate the rate of blastocoel expansion. Although TGF‐α/EGF can elevate cAMP levels in other cell types, TGF‐α, at a concentration that maximally stimulates the rate of blastocoel expansion, did not elevate cAMP in blastocysts. Thus, it was unlikely that elevation of cAMP is the mechanism by which TGF‐α stimulates the rate of blastocoel expansion.
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Blastocyst Transfer
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We analyzed images of human blastocysts obtained from an in vitro culture system we have developed for time-lapse cinematography (TLC). Blastocoel collapse and re-expansion were repeated throughout the blastocyst stage because of disturbance of the trophectoderm (TE) wall. We also found an aberrant “strand phenomenon” within the blastocoel cavity in which the inner cell mass (ICM) ectopically adhered to the opposite TE wall. We also identified that this phenomenon was closely associated with the occurrence of monozygotic twin pregnancies. Monozygotic twinning increases in frequency with day-5 embryo transfers compared with day-2 or day-3 embryo transfers in human ART programs. When the blastocysts eventually escaped through the zona pellucida, we observed two types of hatching (inward and outward). Although the former was always accompanied by blastocoel collapse, this did not occur with outward hatching. We believe that the outward process without blastocoel collapse is more likely to represent the in vivo hatching pattern than the inward process. These results suggest that extended in vitro culture to the late blastocyst stage has a negative impact on human embryonic development.
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Preimplantation or pre-attachment development encompasses the "free"-living period of mammalian embryogenesis, which directs development of the zygote through to the blastocyst stage. Blastocyst formation is essential for implantation, establishment of pregnancy and is a principal determinant of embryo quality prior to embryo transfer. Cavitation (blastocyst formation) is driven by the expression of specific sets of gene products that direct the acquisition of cell polarity within the trophectoderm, which is both the first epithelium of development and the outer cell layer encircling the inner cell mass of the blastocyst. Critical gene families controlling these events include: the E-cadherin-catenin cell adhesion family, the tight junction gene family, the Na/K-ATPase gene family and perhaps the aquaporin gene family. This review will update the roles of each of these gene families in trophectoderm differentiation and blastocyst formation. The current principal hypothesis under investigation is that blastocyst formation is mediated by a trans-trophectoderm ion gradient(s) established, in part, by Na/K-ATPase, which drives the movement of water through aquaporins (AQPs) across the epithelium into the extracellular space of the blastocyst to form the fluid-filled blastocoel. The trophectoderm tight junctional permeability seal regulates the leakage of blastocoel fluid, and also assists in the maintenance of a polarized Na/K-ATPase distribution to the basolateral plasma membrane domain of the mural trophectoderm. The cell-to-cell adhesion provided by the E-cadherin-catenin gene families is required for the establishment of the tight junction seal and the maintenance of the polarized Na/K-ATPase distribution. Blastocyst formation is therefore directly linked with trophectoderm cell differentiation, which arises through fundamental cell biological processes that are associated with the establishment of cell polarity.
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Abstract Study question What is the clinical outcome of transferring a mosaic blastocyst versus a euploid blastocyst in single frozen blastocyst transfer (sFBT) cycles? Summary answer Single mosaic blastocyst transfer has similar clinical outcome to single euploid blastocyst transfer. What is known already Embryonic mosaicism occurs when there are two or more distinct cell lines found in preimplantation embryos derived from IVF. Data from recent studies show that mosaic blastocysts have the potential to implant and can result in healthy live births. As a result, patients now have the option of transferring mosaic blastocyst when they do not have any euploid blastocyst available for transfer. However, the clinical outcome of transferring mosaic blastocyst has not been definitively reported. Thus, a retrospective study was conducted to compare the clinical outcome of mosaic sFBT and euploid sFBT. Study design, size, duration A total of 602 patients underwent frozen blastocyst transfer in Alpha IVF from January to October 2019 and had their blastocysts screened for aneuploidy. These patients were divided into 2 groups: 26 patients with mosaic blastocysts transferred (Group A, age ranged 19–44), and 576 patients with euploid blastocysts transferred (Group B, age ranged 21–44). The mean age of patients from Group A and B were 34.0 and 32.8 respectively (p > 0.05). Participants/materials, setting, methods All samples had their DNA libraries constructed for sequencing using Next Generation Sequencing according to manufacturer’s specification (IonTorrent, USA). All blastocysts were frozen for subsequent sFBT cycle (Cryotech, Japan). All thawed blastocysts for sFBT survived with morphologically intact inner cell mass and trophectoderm cells. The importance of antenatal confirmation of the fetal chromosome status was emphasized in patients from Group A. The clinical outcomes of both groups were analysed and compared. Main results and the role of chance No significant differences were seen in the clinical pregnancy and implantation rate of Group A and B (65.4% vs 63.0%; p > 0.05). The miscarriage rate of Group A and B were 23.5% and 14.0% respectively. Albeit the higher miscarriage rate in Group A, there was no statistical significance between these two groups (p > 0.05). Group A was further divided into two subgroups, Subgroup A1: low risk mosaic blastocyst transfer; Subgroup A2: high risk mosaic blastocyst transfer. In the comparison of Group A subgroups, the clinical pregnancy and implantation of Group A1 is higher than Group A2 (76.9% vs 44.4%). In addition, the miscarriage rate of Group A1 and A2 were 23.1% and 0.0% respectively. Interestingly, there was no statistical significance in clinical pregnancy rate, implantation rate and miscarriage rate between these two subgroups. Limitations, reasons for caution This is a retrospective study and the sample size was comparatively smaller in the mosaic blastocyst transfer group than the euploid blastocyst transfer group. Further studies with a larger sample size should be carried out to ascertain the clinical outcome. Wider implications of the findings: Single mosaic blastocyst transfer has similar clinical outcome to single euploid blastocyst transfer. Thus, mosaic blastocyst can be considered for transfer when no euploid blastocyst are available. Nevertheless, stringent antenatal surveillance for chromosomal abnormalities to confirm the chromosomal status of the fetus must be followed. Trial registration number Not applicable
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Abstract Objective To determine whether the morphologic parameters of euploid blastocyst influence the live birth rate (LBR) following single frozen-thawed embryo transfer (FET) cycles? Methods A retrospective cohort analysis involving autologous single FET cycles after next generation sequencing (NGS) based preimplantation genetic testing for aneuploidy (PGT-A) by a large in vitro fertilization (IVF) center that was performed from June 2017 to September 2019.Women were divided into three age groups (< 30, 30–34 and ≥ 35 years old). The primary outcome measure was LBR. Outcomes were compared between different blastocyst quality (Good, Average and Poor), inner cell mass (ICM) grade (A and B), and trophectoderm (TE) grade (A, B and C). Results A total of 232 FET cycles were included, the live birth rate was 48.28%. In the youngest group (< 30 years old, n = 86), LBR were compared between cycles with various blastocyst quality (72.22% for good quality, 54.55% for average quality and 34.78% for poor quality; P = 0.019), ICM grade (70.59% for grade A and 42.03% for grade B; P = 0.035) and TE grade (85.71% for grade A,57.58% for grade B and 34.78 for grade C; P = 0.015). Nevertheless, either in the 30–34 years group (n = 99) or in the oldest group (≥ 35years, n = 47), LBR were also comparable between these subgroups, no significant difference was showed in blastocyst morphologic parameters and LBR ( P > 0.05). Furthermore, in the similarly graded euploid blastocysts, there was also no statistical significance in LBR among different age subgroups ( P > 0.05). Conclusions In women ≥ 30 years old, euploid blastocyst quality was not associated with the LBR in FET cycles, highlights the development competence of poor-quality euploid blastocysts.
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Abstract The influence of the location of a trophectoderm biopsy in human blastocysts on the development of those blastocysts has not yet been investigated. In our prospective study (n=92), our multivariate logistic regression analysis indicated that blastocoel development was influenced by the location of the trophectoderm biopsy (p=0.049) and by the type of human blastocyst used (fresh or thawed) (p=0.037), regardless of the patient’s age (p=0.507) and the number of days for the human blastocyst in the pretrophectoderm biopsy (p=0.239). Therefore, when a trophectoderm biopsy is close to the inner cell mass (ICM) in human blastocysts, it improves the progress of blastocoel development. Clinical evidence suggests that the progress of blastocoel development is a predictor of clinical outcomes after single blastocyst transfer. Therefore, when the trophectoderm biopsy is done from near the ICM, improvement of clinical outcomes after single blastocyst transfer may be expected.
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