Full developmental potential of mammalian preimplantation embryos is maintained after imaging using a spinning-disk confocal microscope

Abstract Advanced maternal age (AMA) is known to reduce fertility, increases aneuploidy in oocytes and early embryos and leads to adverse developmental consequences which may associate with offspring lifetime health risks. However, investigating underlying effects of AMA on embryo developmental potential is confounded by the inherent senescence present in maternal body systems further affecting reproductive success. Here, we describe a new model for the analysis of early developmental mechanisms underlying AMA by the derivation and characterisation of mouse embryonic stem cell (mESC-like) lines from naturally conceived embryos. Young (7–8 weeks) and Old (7–8 months) C57BL/6 female mice were mated with young males. Preimplantation embryos from Old dams displayed developmental retardation in blastocyst morphogenesis. mESC lines established from these blastocysts using conventional techniques revealed differences in genetic, cellular and molecular criteria conserved over several passages in the standardised medium. mESCs from embryos from AMA dams displayed increased incidence of aneuploidy following Giemsa karyotyping compared with those from Young dams. Moreover, AMA caused an altered pattern of expression of pluripotency markers (Sox2, OCT4) in mESCs. AMA further diminished mESC survival and proliferation and reduced the expression of cell proliferation marker, Ki-67. These changes coincided with altered expression of the epigenetic marker, Dnmt3a and other developmental regulators in a sex-dependent manner. Collectively, our data demonstrate the feasibility to utilise mESCs to reveal developmental mechanisms underlying AMA in the absence of maternal senescence and with reduced animal use.

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Intracellular localisation of platelet-activating factor during mammalian embryo development in vitro: a comparison of cattle, mouse and human

Reproduction Fertility and Development ◽

10.1071/rd18146 ◽

2019 ◽

Vol 31 (4) ◽

pp. 658

Author(s):

L. T. M. Vandenberghe ◽

B. Heindryckx ◽

K. Smits ◽

M. Popovic ◽

K. Szymanska ◽

...

Keyword(s):

Platelet Activating Factor ◽

Preimplantation Embryos ◽

Mammalian Embryo ◽

Developmental Potential ◽

Nuclear Envelope Breakdown ◽

Cytosolic Phospholipase ◽

Carrier Molecule ◽

Development In Vitro ◽

Intracellular Localisation

Platelet-activating factor (PAF) is a well-known marker for embryo quality and viability. For the first time, we describe an intracellular localisation of PAF in oocytes and embryos of cattle, mice and humans. We showed that PAF is represented in the nucleus, a signal that was lost upon nuclear envelope breakdown. This process was confirmed by treating the embryos with nocodazole, a spindle-disrupting agent that, as such, arrests the embryo in mitosis, and by microinjecting a PAF-specific antibody in bovine MII oocytes. The latter resulted in the absence of nuclear PAF in the pronuclei of the zygote and reduced further developmental potential. Previous research indicates that PAF is released and taken up from the culture medium by preimplantation embryos invitro, in which bovine serum albumin (BSA) serves as a crucial carrier molecule. In the present study we demonstrated that nuclear PAF does not originate from an extracellular source because embryos cultured in polyvinylpyrrolidone or BSA showed similar levels of PAF in their nuclei. Instead, our experiments indicate that cytosolic phospholipase A2 (cPLA2) is likely to be involved in the intracellular production of PAF, because treatment with arachidonyl trifluoromethyl ketone (AACOCF3), a specific cPLA2 inhibitor, clearly lowered PAF levels in the nuclei of bovine embryos.

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Three-Dimensional Live Imaging of Bovine Preimplantation Embryos: A New Method for IVF Embryo Evaluation

Frontiers in Veterinary Science ◽

10.3389/fvets.2021.639249 ◽

2021 ◽

Vol 8 ◽

Author(s):

Yasumitsu Masuda ◽

Ryo Hasebe ◽

Yasushi Kuromi ◽

Masayoshi Kobayashi ◽

Kanako Urataki ◽

...

Keyword(s):

Embryo Transfer ◽

Inner Cell Mass ◽

Cell Mass ◽

Three Dimensional ◽

Preimplantation Embryos ◽

Infrared Light ◽

Bovine Embryo ◽

Bovine Embryos ◽

Cell Stage ◽

Developmental Potential

Conception rates for transferred bovine embryos are lower than those for artificial insemination. Embryo transfer (ET) is widely used in cattle but many of the transferred embryos fail to develop, thus, a more effective method for selecting bovine embryos suitable for ET is required. To evaluate the developmental potential of bovine preimplantation embryos (2-cell stage embryos and blastocysts), we have used the non-invasive method of optical coherence tomography (OCT) to obtain live images. The images were used to evaluate 22 parameters of blastocysts, such as the volume of the inner cell mass and the thicknesses of the trophectoderm (TE). Bovine embryos were obtained by in vitro fertilization (IVF) of the cumulus-oocyte complexes aspirated by ovum pick-up from Japanese Black cattle. The quality of the blastocysts was examined under an inverted microscope and all were confirmed to be Code1 according to the International Embryo Transfer Society standards for embryo evaluation. The OCT images of embryos were taken at the 2-cell and blastocyst stages prior to the transfer. In OCT, the embryos were irradiated with near-infrared light for a few minutes to capture three-dimensional images. Nuclei of the 2-cell stage embryos were clearly observed by OCT, and polynuclear cells at the 2-cell stage were also clearly found. With OCT, we were able to observe embryos at the blastocyst stage and evaluate their parameters. The conception rate following OCT (15/30; 50%) is typical for ETs and no newborn calves showed neonatal overgrowth or died, indicating that the OCT did not adversely affect the ET. A principal components analysis was unable to identify the parameters associated with successful pregnancy, while by using hierarchical clustering analysis, TE volume has been suggested to be one of the parameters for the evaluation of bovine embryo. The present results show that OCT imaging can be used to investigate time-dependent changes of IVF embryos. With further improvements, it should be useful for selecting high-quality embryos for transfer.

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Rapid Frequency-Domain FLIM Spinning Disk Confocal Microscope: Lifetime Resolution, Image Improvement and Wavelet Analysis

Journal of Fluorescence ◽

10.1007/s10895-008-0332-3 ◽

2008 ◽

Vol 18 (5) ◽

pp. 929-942 ◽

Cited By ~ 41

Author(s):

Chittanon Buranachai ◽

Daichi Kamiyama ◽

Akira Chiba ◽

Benjamin D. Williams ◽

Robert M. Clegg

Keyword(s):

Wavelet Analysis ◽

Frequency Domain ◽

Confocal Microscope ◽

Resolution Image ◽

Spinning Disk ◽

Image Improvement

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P–516 Evidence for self-correction in preimplantation embryos by comparative molecular karyotyping of blastocoele fluid, trophectoderm and inner cell mass

Human Reproduction ◽

10.1093/humrep/deab130.515 ◽

2021 ◽

Vol 36 (Supplement_1) ◽

Author(s):

D Zhigalina ◽

N Skryabin ◽

O Kanbekova ◽

V Artyukhova ◽

A Svetlakov ◽

...

Keyword(s):

Inner Cell Mass ◽

Cell Mass ◽

Morphological Characteristics ◽

Preimplantation Embryos ◽

Molecular Karyotyping ◽

Embryonic Cells ◽

Developmental Potential ◽

Positive Correlation ◽

Registration Number ◽

Molecular Karyotype

Abstract Study question Does the molecular karyotype of the cell-free DNA (cfDNA) from the blastocyst fluid (BF) can predict the efficiency of self-correction of karyotype of preimplantation embryo? Summary answer Detection of aneuploidies in the BF potentially can point out on effective self-correction of blastocyst karyotype and consequently on high developmental potential of mosaic embryos. What is known already Correction of aneuploidies in the preimplantation embryos can be provided by several mechanisms, including apoptosis. The predominant death of aneuploid cells was demonstrated in mouse embryos (Bolton, 2016). A positive correlation was also shown between the concentration of cfDNA from the BF of human blastocyst and the morphology of the embryo, as well as between the activity of caspase–3 and the concentration of cfDNA (Rule, 2018). The incidence of failed amplification after WGA being significantly higher among euploid blastocysts (Magli, 2019). The capacity of abnormal cells extruding into the BF would be related to the embryo development potential (Gianaroli, 2019). Study design, size, duration This is a prospective observational study of thirty-one Day 5 human blastocysts. Cryopreserved blastocysts were received after treatment cycles at the IVF Center with informed consent obtained from couples. The average age of 15 women was 32.25±5 years. The morphological characteristics of blastocysts were estimated in accordance with the Gardner classification (Gardner, Schoolcraft, 1999). The procedure of BF aspiration and trophectoderm (TE) and ICM cells separation of the blastocysts was previously described (Tsuiko, 2018). Participants/materials, setting, methods WGA was performed by PicoPLEX kit (Rubicon Genomics, USA) or REPLI-g Mini kit (Qiagen) according to manufacturer’s protocols. The DNA of the BF, ICM and TE were analyzed separately using cCGH, aCGH and NGS. SurePrint G3 Human CGH Microarrays (8x60K, Agilent Technologies) were used according to the manufacturer’s recommendations. Image analysis was done using ISIS (v.5.5) (Metasystems) and Agilent CytoGenomics Software (v.3). VeriSeq™ PGS Kit - MiSeq® System (Illumina) was used for NGS. Main results and the role of chance Molecular karyotypes of all three samples - BF, ICM and TE, were obtained for 23 (74.2%) blastocysts. A correlation between the woman’s age and the number of aneuploidies in cfDNA (p = 0.0009) was found. A positive correlation may indicate that the number of aneuploidies in the embryonic cells increases with the age of a woman, however, the embryonic karyotype undergoes self-correcting through the elimination of aneuploid cells. It was noted that well-developing blastocysts (groups 4–5, according to Gardner’s classification) had fewer aneuploidies in ICM (p = 0.0141) and TE (p = 0.0436). In contrast, there was a tendency to an increase in the number of aneuploidies in the BF during blastocysts transition from stage 3 to 5 (p = 0.3542). We assessed the relationship between the number of aneuploidies in groups of blastocysts with different characteristics of ICM (groups “A” and “B” according to Gardner’s classification). These groups significantly differ in the number of aneuploidies in cfDNA (p = 0.0352), although the statistically significant differences between the number of aneuploidies in ICM (p = 0.5992) and in TE (p = 0.5934) was not detected. Thus, higher-quality embryos in terms of ICM morphology contain more abnormalities in the BF, since in this group the elimination of aneuploid cells is more efficient. Limitations, reasons for caution The number of embryos is limited in this study. More comprehensive studies are required to confirm the observed tendency. Wider implications of the findings: Aneuploid cells elimination can be a cause of increasing cfDNA concentration in the BF, which may be a marker of the viability of mosaic embryos when it is necessary to decide on mosaic embryo transfer. This study was supported by the RFBR (15–04–08265) and by the RSF (20–74–00064). Trial registration number Not applicable

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Replication stress limits developmental potential of human preimplantation embryos

Fertility and Sterility ◽

10.1016/j.fertnstert.2019.07.454 ◽

2019 ◽

Vol 112 (3) ◽

pp. e128

Author(s):

Katherine L. Palmerola ◽

Eric J. Forman ◽

Rogerio A. Lobo ◽

Dieter M. Egli

Keyword(s):

Replication Stress ◽

Preimplantation Embryos ◽

Developmental Potential ◽

Human Preimplantation Embryos

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Anti-silencing factor 1A is associated with genome stability maintenance of mouse preimplantation embryos†

Biology of Reproduction ◽

10.1093/biolre/ioaa001 ◽

2020 ◽

Vol 102 (4) ◽

pp. 817-827

Author(s):

Kai Deng ◽

Wanyou Feng ◽

Xiaohua Liu ◽

Xiaoping Su ◽

Erwei Zuo ◽

...

Keyword(s):

Embryonic Development ◽

Genome Stability ◽

Mouse Embryos ◽

Preimplantation Embryos ◽

End Joining ◽

Dna Damages ◽

Developmental Potential ◽

Damage Repair ◽

Non Homologous End Joining ◽

Early Mouse

Abstract Genome stability is critical for the normal development of preimplantation embryos, as DNA damages may result in mutation and even embryo lethality. Anti-silencing factor 1A (ASF1A) is a histone chaperone and enriched in the MII oocytes as a maternal factor, which may be associated with the maintenance of genome stability. Thus, this study was undertaken to explore the role of ASF1A in maintaining the genome stability of early mouse embryos. The ASF1A expressed in the preimplantation embryos and displayed a dynamic pattern throughout the early embryonic development. Inhibition of ASF1A expression decreased embryonic development and increased DNA damages. Overexpression of ASF1A improved the developmental potential and decreased DNA damages. When 293T cells that had been integrated with RGS-NHEJ were co-transfected with plasmids of pcDNA3.1-ASF1A, gRNA-NHEJ, and hCas9, less cells expressed eGFP, indicating that non-homologous end joining was reduced by ASF1A. When 293T cells were co-transfected with plasmids of HR-donor, gRNA-HR, hCas9, and pcDNA3.1-ASF1A, more cells expressed eGFP, indicating that homologous recombination (HR) was enhanced by ASF1A. These results indicate that ASF1A may be associated with the genome stability maintenance of early mouse embryos and this action may be mediated by promoting DNA damage repair through HR pathway.

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Total ablation of paternal accessory sex glands curtails developmental potential in preimplantation embryos in the golden hamster

Anatomy and Embryology ◽

10.1007/s004290100186 ◽

2001 ◽

Vol 204 (2) ◽

pp. 117-122 ◽

Cited By ~ 13

Author(s):

O. C. Chan ◽

P. H. Chow ◽

W. S. O

Keyword(s):

Golden Hamster ◽

Preimplantation Embryos ◽

Developmental Potential ◽

Accessory Sex Glands

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Developmental and molecular correlates of bovine preimplantation embryos

Reproduction ◽

10.1530/rep.1.01021 ◽

2006 ◽

Vol 131 (5) ◽

pp. 895-904 ◽

Cited By ~ 60

Author(s):

Hakan Sagirkaya ◽

Muge Misirlioglu ◽

Abdullah Kaya ◽

Neal L First ◽

John J Parrish ◽

...

Keyword(s):

Gene Expression ◽

Dna Methyltransferase ◽

Expression Profiles ◽

Expression Patterns ◽

Blastocyst Stage ◽

Culture Conditions ◽

Preimplantation Embryos ◽

Developmental Potential

Expression of embryonic genes is altered in different culture conditions, which influence developmental potential both during preimplantation and fetal development. The objective of this study was to define the effects of culture conditions on: bovine embryonic development to blastocyst stage, blastocyst cell number, apoptosis and expression patterns of a panel of developmentally important genes. Bovine embryos were culturedin vitroin three culture media containing amino acids, namely potassium simplex optimization medium (KSOMaa), Charles Rosenkrans 1 (CR1aa) and synthetic oviductal fluid (SOFaa). Apoptosis in blastocysts was determined by TUNEL assay and expression profiles of developmentally important genes were assayed by real-time PCR.In vivo-produced bovine blastocysts were used as controls for experiments determining gene expression patterns. While the cleavage rates did not differ, embryos cultured in SOFaa had higher rates of development to blastocyst stage (P< 0.05). Mean cell numbers and percentages of apoptotic cells per blastocyst did not differ among the groups. Expression of the heat shock protein 70 (Hsp70) gene was significantly up-regulated in both CR1aa and KSOMaa when compared with SOFaa (P< 0.001). DNA methyltransferase 3a (Dnmt3a) expression was higher in embryos cultured in CR1aa than in those cultured in SOFaa (P< 0.001). Expression of interferon tau (IF-τ) and insulin-like growth factor II receptor (Igf-2r) genes was significantly up-regulated in KSOMaa when compared with CR1aa (P< 0.001). Gene expression did not differ betweenin vivo-derived blastocysts and theirin vitro-derived counterparts. In conclusion, SOFaa supports higher development to blastocyst stage than KSOMaa and CR1aa, and the culture conditions influence gene expression.

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