What Drives Embryo Development? Chromosomal Normality or Mitochondria?

Objective. To report the arrest of euploid embryos with high mtDNA content. Design. A report of 2 cases. Setting. Private fertility clinic. Patients. 2 patients, 45 and 40 years old undergoing IVF treatment. Interventions. Mature oocytes were collected and vitrified from two ovarian stimulations. Postthaw, survived mature oocytes underwent fertilization by intracytoplasmic sperm injection (ICSI). Preimplantation genetic screening (PGS) and mitochondrial DNA (mtDNA) copy number were done using next generation sequencing (NGS). The only normal embryo among the all-biopsied embryos had the highest “Mitoscore” value and was the only arrested embryo in both cases. Therefore, the embryo transfer was cancelled. Main Outcome Measures. Postthaw survival and fertilization rate, embryo euploidy, mtDNA copy number, and embryo development. Results. In both patients, after PGS only 1 embryo was euploid. Both embryos had the highest mtDNA copy number from all tested embryos and both embryos were arrested on further development. Conclusions. These cases clearly demonstrate the lack of correlation between mtDNA value (Mitoscore) and chromosomal status of embryo.

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86 Effect of invitro culture conditions on mitochondria functions in mouse embryos

Reproduction Fertility and Development ◽

10.1071/rdv32n2ab86 ◽

2020 ◽

Vol 32 (2) ◽

pp. 169

Author(s):

M. Czernik ◽

D. Winiarczyk ◽

S. Sampino ◽

P. Greda ◽

J. A. Modlinski ◽

...

Keyword(s):

Embryo Development ◽

Copy Number ◽

Energy Demand ◽

Mitochondrial Membrane ◽

Mitochondrial Dynamics ◽

Culture Conditions ◽

Mouse Embryos ◽

Mtdna Copy Number ◽

Mitochondrial Functions ◽

Mitochondria Functions

Mitochondria provide the energy for oocyte maturation, fertilisation, and embryo formation via oxidative phosphorylation. Consequently, any adverse influence on mitochondrial function may negatively affect the development of pre-implantation embryos especially because there is no mitochondrial DNA (mtDNA) replication until post-implantation. Studies in the field of mitochondrial dynamics have identified an intriguing link between energy demand/supply balance and mitochondrial architecture, which may suggest that inappropriate culture conditions may inhibit mitochondrial functions, which may negatively affect embryo development. We wanted to check whether invitro culture (IVC) conditions of mouse embryos affect mitochondrial functionality. The IVC as well as naturally matted (NM) mouse embryos at the 2-cell and blastocyst stage were subjected to mitochondrial analysis (distribution, organisation, and mitochondrial membrane potential), and expression of mRNA and proteins involved in regulation of mitochondria functions, as well as number of mtDNA copies, were evaluated. Significance level was set at 0.05. We observed that the mitochondria in 2-cell IVC embryos were less numerous and localised mainly in the pericortical region of the cytoplasm, whereas mitochondria in NM embryos were numerous and homogeneously distributed in both blastomeres. Drastic differences were observed in blastocysts. Mitochondria in the IVC group were fragmented, rounded, and aggregated mainly in the perinuclear region of the cells, whereas mitochondria of NM blastocysts were numerous and created an elongated mitochondrial network along the cells. Time-lapse analysis showed reduced mitochondrial and mitochondrial membrane activity in IVC blastocysts. Moreover, our results indicate the IVC group had reduced mRNA expression of mitofusin 1, mitofusin 2, and optic atrophy 1 responsible for mitochondrial fusion. Additionally, mtDNA copy number for IVC blastocysts (398 887.45±30 608.65) was significantly lower than that of NM blastocysts (593 367.12±66 540.32; P<0.02). Furthermore, no significant differences were found in mtDNA copy number of IVC 2-cell embryos when compared with NM embryos. The results obtained clearly showed that IVC conditions affect proper mitochondrial functionality and hence embryo development.

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Copy number alterations in cancer: detection and precision medicine

BIO Web of Conferences ◽

10.1051/bioconf/20214102005 ◽

2021 ◽

Vol 41 ◽

pp. 02005

Author(s):

Arief Gusnanto

Keyword(s):

Next Generation Sequencing ◽

Precision Medicine ◽

Cancer Patients ◽

Cancer Detection ◽

Copy Number ◽

Copy Number Alterations ◽

Genomic Alterations ◽

Genomic Markers ◽

Next Generation Sequencing Ngs ◽

Generation Sequencing

Copy number alterations (CNAs) are genomic alterations where some regions exhibit more or less copy number than the normal two copies. In this talk, I will describe two ideas: (1) how CNAs are estimated from data generated by next generation sequencing (NGS) and what steps are required to make the data interpretable, (2) how the CNA can be utilised for precision medicine in terms of prediction of tumour subtypes and prediction of cancer patients’ survival. If time permits, I will also discuss how to estimate genomic markers from CNA profile across cancer patients.

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Preimplantation Genetic Diagnosis

10.1093/med/9780199329007.003.0022 ◽

2018 ◽

Author(s):

R. J McKinlay Gardner ◽

David J Amor

Keyword(s):

Older Women ◽

Preimplantation Genetic Diagnosis ◽

Genetic Diagnosis ◽

Preimplantation Genetic Screening ◽

Normal Embryo ◽

Vitro Fertilization ◽

Blastocyst Biopsy ◽

Time Stage ◽

Generation Sequencing

Preimplantation genetic diagnosis allows recognition of a genetically abnormal embryo in the laboratory, and enables the choice, in principle, of selecting a normal embryo for transfer to the uterus. The methodologies are outlined in this chapter, noting the move toward day-5 blastocyst biopsy as the preferred time/stage. Next-generation sequencing is also discussed. The distinction is made between targeted diagnosis, as for example in the setting of a parental rearrangement, and preimplantation genetic screening, which may be offered to older women or those who, in any event, need recourse to in vitro fertilization. The improved diagnostic precision due to molecular methodology is noted.

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6 THE EFFECTS OF DEPLETING DONOR CELL MITOCHONDRIAL DNA ON CATTLE EMBRYOS DERIVED FROM SOMATIC CELL NUCLEAR TRANSFER

Reproduction Fertility and Development ◽

10.1071/rdv28n2ab6 ◽

2016 ◽

Vol 28 (2) ◽

pp. 132 ◽

Cited By ~ 1

Author(s):

K. Srirattana ◽

J. C. St. John

Keyword(s):

Mitochondrial Dna ◽

Somatic Cell ◽

Embryo Development ◽

Copy Number ◽

Somatic Cell Nuclear Transfer ◽

Nuclear Transfer ◽

Trichostatin A ◽

Donor Cell ◽

Mtdna Copy Number ◽

Donor Cells

Although somatic cell nuclear transfer (SCNT) is a valuable tool for producing animals for agricultural and research purposes, the resultant mixing of mitochondrial DNA (mtDNA) from the donor cell and recipient oocyte (heteroplasmy) affects embryo development and offspring survival and health. The aim of this study was to determine the effects of depleting donor cells of their mtDNA before SCNT on embryo development. mtDNA was depleted from cattle fibroblasts using 2′,3′-dideoxycytidine. mtDNA copy number in cells depleted for 30 days (0.85 ± 0.05) was significantly decreased when compared with nondepleted cells (150.12 ± 29.90; P < 0.0001, ANOVA). Moreover, mtDNA copy number in depleted cells could not be replenished after depletion for 30 days. Depleted cells and nondepleted cells were used as donor cells for SCNT. Somatic cell nuclear transfer embryos were produced by electrofusion of a single donor cell with an enucleated cow oocyte. Reconstructed oocytes were chemically activated and cultured for 7 days (nontreated embryos). Another cohort of embryos was treated with Trichostatin A (TSA), to enhance reprogramming, by activating reconstructed oocytes and culturing them in the presence of 50 nM TSA for up to 10 h. The embryos were then cultured in the absence of TSA. In nontreated groups, the fusion rates of depleted cells (78.0 ± 0.8%) were significantly lower than those of nondepleted cells (92.1 ± 1.4%; P < 0.05). No positive effect on fusion rates was found after TSA treatment. The blastocyst rate for SCNT embryos derived from depleted cells (18.7 ± 4.9%) was significantly lower than the nondepleted group (32.5 ± 3.1%; P < 0.05). Trichostatin A treatment increased blastocyst rates for SCNT embryos derived from depleted cells (32.5 ± 5.3%) to levels equivalent to those of nondepleted cells but did not have any beneficial effect on SCNT embryos derived from nondepleted cells. We have analysed blastocysts for the presence of donor cell mtDNA by high resolution melting analysis. Four out of 10 SCNT blastocysts derived from nondepleted cells were heteroplasmic, whereas others had no donor cell mtDNA. However, all 10 analysed SCNT blastocysts derived from depleted cells were homoplasmic as they harboured only oocyte mtDNA. From RNA sequencing results, TSA treatment of SCNT blastocysts derived from depleted cells increased the expression of key developmental transcription regulators and decreased expression of the mtDNA-specific replication factors, which is essential for embryo development. In conclusion, homoplasmic SCNT embryos were successfully produced by using mtDNA depleted donor cells. Trichostatin A treatment enhanced nuclear reprogramming efficiency in SCNT embryos derived from depleted cells. This work was supported by MitoStock Pty. Ltd., Australia.

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