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A systematic analysis of the suitability of preimplantation genetic diagnosis for mitochondrial diseases in a heteroplasmic mitochondrial mouse model

Jitesh Neupane, Mado Vandewoestyne UGent, Björn Heindryckx UGent, Sabitri Ghimire, Yuechao Lu UGent, Chen Qian UGent, Sylvie Lierman, Rudy Van Coster UGent, Jan Gerris UGent, Tom Deroo, et al. (2014) HUMAN REPRODUCTION. 29(4). p.852-859
abstract
Study question: What is the reliability of preimplantation genetic diagnosis (PGD) based on polar body (PB), blastomere or trophectoderm (TE) analysis in a heteroplasmic mitochondrial mouse model? Summary answer: The reliability ofPGDto determine the level of mitochondrialDNA(mtDNA) heteroplasmy is questionable based on either the first or second PB analysis; however, PGD based on blastomere or TE analysis seems more reliable. What is known already: PGD has been suggested as a technique to determine the level of mtDNA heteroplasmy in oocytes and embryos to avoid the transmission of heritable mtDNA disorders. A strong correlation between first PBs and oocytes and between second PBs and zygotes was reported in mice but is controversial in humans. So far, the levels of mtDNA heteroplasmy in first PBs, second PBs and their corresponding oocytes, zygotes and blastomeres, TE and blastocysts have not been analysed within the same embryo. Study design, size and duration: We explored the suitability of PGD by comparing the level of mtDNA heteroplasmy between first PBs and metaphase II (MII) oocytes (n ¼ 33), between first PBs, second PBs and zygotes (n ¼ 30), and between first PBs, second PBs and their corresponding blastomeres of 2- (n ¼ 10), 4- (n ¼ 10) and 8-cell embryos (n ¼ 11). Levels of mtDNA heteroplasmy in second PBs (n ¼ 20), single blastomeres from 8-cell embryos (n ¼ 20), TE (n ¼ 20) and blastocysts (n ¼ 20) were also compared. Participants/materials, setting,methods: Heteroplasmic mice (BALB/cOlaHsd), containingmtDNAmixtures ofBALB/cByJ and NZB/OlaHsd,were used in this study. The first PBs were biopsied from in vivo matured MII oocytes. The ooplasm was then subjected to ICSI. After fertilization, second PBs were biopsied and zygotes were cultured to recover individual blastomeres from 2-, 4- and 8-cell embryos. Similarly, second PBs were biopsied from in vivo fertilized zygotes and single blastomeres were biopsied from 8-cell stage embryos. The remaining embryowas cultured until the blastocyst stage to isolate TE cells. Polymerase chain reaction followedby restriction fragment length polymorphism was performed to measure the level of mtDNA heteroplasmy in individual samples. Main results and the role of chance: Modest correlations and wide prediction interval [PI at 95% confidence interval (CI)] were observed in the level ofmtDNAheteroplasmy between first PBs and their corresponding MII oocytes (r2 ¼ 0.56; PI ¼ 45.96%) and zygotes (r2 ¼ 0.69; PI ¼ 37.07%). The modest correlations and wide PI were observed between second PBs and their corresponding zygotes (r2 ¼ 0.65; PI ¼ 39.69%), single blastomeres (r2 ¼ 0.42; PI ¼ 48.04%), TE (r2 ¼ 0.26; PI ¼ 54.79%) and whole blastocysts (r2 ¼ 0.40; PI ¼ 57.48%). A strong correlation with a narrow PI was observed among individual blastomeres of 2-, 4- and 8-cell stage embryos (r2 ¼ 0.92; PI ¼ 11.73%, r2 ¼ 0.86; PI ¼ 18.85% and r2 ¼ 0.85; PI ¼ 21.42%, respectively), and also between TE and whole blastocysts (r2 ¼ 0.90; PI ¼ 23.58%). Moreover, single blastomeres from 8-cell stage embryos showed a close correlation and an intermediate PI with corresponding TE cells (r2 ¼ 0.81; PI ¼ 28.15%) and blastocysts (r2 ¼ 0.76; PI ¼ 36.43%). Limitations, reasons for caution: These results in a heteroplasmic mitochondrial mouse model should be further verified in patients with mtDNA disorders to explore the reliability of PGD. Wider implications of the findings: To avoid the transmission of heritable mtDNA disorders, PGD techniques should accurately determine the level of heteroplasmy in biopsied cells faithfully representing the heteroplasmic load in oocytes and preimplantation embryos. Unlike previous PGD studies in mice, our results accord with PGD results for mitochondrial disorders in humans, and question the reliability of PGD using different stages of embryonic development. Study funding/competing interest(s): This work was funded by a doctoral grant provided by the Special Research Fund (BOF) to J.N. (grant number, 01D05611) and a postdoctoral grant provided by Research Foundation-Flanders (FWO) to M.V. (grant number, 1248413N). P.S. is holder of a fundamental clinical research mandate by FWO-Vlaanderen (Flemish fund for scientific research). Trial registration number: Not applicable
Please use this url to cite or link to this publication:
author
organization
year
type
journalArticle (original)
publication status
published
subject
keyword
preimplantation embryos, preimplantation genetic diagnosis, mtDNA heteroplasmy, mitochondrial DNA, mtDNA disorders, MUTATION LOAD, POLAR BODIES, DNA, MTDNA, SEGREGATION, OOCYTES, PGD, TRANSMISSION, INHERITANCE, BLASTOMERES
journal title
HUMAN REPRODUCTION
Hum. Reprod.
volume
29
issue
4
pages
852 - 859
Web of Science type
Article
Web of Science id
000333051200023
JCR category
OBSTETRICS & GYNECOLOGY
JCR impact factor
4.569 (2014)
JCR rank
5/79 (2014)
JCR quartile
1 (2014)
ISSN
0268-1161
DOI
10.1093/humrep/deu016
language
English
UGent publication?
yes
classification
A1
copyright statement
I have transferred the copyright for this publication to the publisher
id
4382473
handle
http://hdl.handle.net/1854/LU-4382473
date created
2014-05-13 14:15:18
date last changed
2016-12-19 15:38:32
@article{4382473,
  abstract     = {Study question: What is the reliability of preimplantation genetic diagnosis (PGD) based on polar body (PB), blastomere or trophectoderm (TE) analysis in a heteroplasmic mitochondrial mouse model?
Summary answer: The reliability ofPGDto determine the level of mitochondrialDNA(mtDNA) heteroplasmy is questionable based on either the first or second PB analysis; however, PGD based on blastomere or TE analysis seems more reliable.
What is known already: PGD has been suggested as a technique to determine the level of mtDNA heteroplasmy in oocytes and embryos to avoid the transmission of heritable mtDNA disorders. A strong correlation between first PBs and oocytes and between second PBs and zygotes was reported in mice but is controversial in humans. So far, the levels of mtDNA heteroplasmy in first PBs, second PBs and their corresponding oocytes, zygotes and blastomeres, TE and blastocysts have not been analysed within the same embryo.
Study design, size and duration: We explored the suitability of PGD by comparing the level of mtDNA heteroplasmy between first PBs and metaphase II (MII) oocytes (n {\textonequarter} 33), between first PBs, second PBs and zygotes (n {\textonequarter} 30), and between first PBs, second PBs and their corresponding blastomeres of 2- (n {\textonequarter} 10), 4- (n {\textonequarter} 10) and 8-cell embryos (n {\textonequarter} 11). Levels of mtDNA heteroplasmy in second PBs (n {\textonequarter} 20), single blastomeres from 8-cell embryos (n {\textonequarter} 20), TE (n {\textonequarter} 20) and blastocysts (n {\textonequarter} 20) were also compared.
Participants/materials, setting,methods: Heteroplasmic mice (BALB/cOlaHsd), containingmtDNAmixtures ofBALB/cByJ and NZB/OlaHsd,were used in this study. The first PBs were biopsied from in vivo matured MII oocytes. The ooplasm was then subjected to ICSI. After fertilization, second PBs were biopsied and zygotes were cultured to recover individual blastomeres from 2-, 4- and 8-cell embryos. Similarly, second PBs were biopsied from in vivo fertilized zygotes and single blastomeres were biopsied from 8-cell stage embryos. The remaining embryowas cultured until the blastocyst stage to isolate TE cells. Polymerase chain reaction followedby restriction fragment length polymorphism was performed to measure the level of mtDNA heteroplasmy in individual samples.
Main results and the role of chance: Modest correlations and wide prediction interval [PI at 95\% confidence interval (CI)] were observed in the level ofmtDNAheteroplasmy between first PBs and their corresponding MII oocytes (r2 {\textonequarter} 0.56; PI {\textonequarter} 45.96\%) and zygotes (r2 {\textonequarter} 0.69; PI {\textonequarter} 37.07\%). The modest correlations and wide PI were observed between second PBs and their corresponding zygotes (r2 {\textonequarter} 0.65; PI {\textonequarter} 39.69\%), single blastomeres (r2 {\textonequarter} 0.42; PI {\textonequarter} 48.04\%), TE (r2 {\textonequarter} 0.26; PI {\textonequarter} 54.79\%) and whole blastocysts (r2 {\textonequarter} 0.40; PI {\textonequarter} 57.48\%). A strong correlation with a narrow PI was observed among individual blastomeres of 2-, 4- and 8-cell stage embryos (r2 {\textonequarter} 0.92; PI {\textonequarter} 11.73\%, r2 {\textonequarter} 0.86; PI {\textonequarter} 18.85\% and r2 {\textonequarter} 0.85; PI {\textonequarter} 21.42\%, respectively), and also between TE and whole blastocysts (r2 {\textonequarter} 0.90; PI {\textonequarter} 23.58\%). Moreover, single blastomeres from 8-cell stage embryos showed a close correlation and an intermediate PI with corresponding TE cells (r2 {\textonequarter} 0.81; PI {\textonequarter} 28.15\%) and blastocysts (r2 {\textonequarter} 0.76; PI {\textonequarter} 36.43\%).
Limitations, reasons for caution: These results in a heteroplasmic mitochondrial mouse model should be further verified in patients with mtDNA disorders to explore the reliability of PGD.
Wider implications of the findings: To avoid the transmission of heritable mtDNA disorders, PGD techniques should accurately determine the level of heteroplasmy in biopsied cells faithfully representing the heteroplasmic load in oocytes and preimplantation embryos. Unlike previous PGD studies in mice, our results accord with PGD results for mitochondrial disorders in humans, and question the reliability of PGD using different stages of embryonic development.
Study funding/competing interest(s): This work was funded by a doctoral grant provided by the Special Research Fund (BOF) to J.N. (grant number, 01D05611) and a postdoctoral grant provided by Research Foundation-Flanders (FWO) to M.V. (grant number, 1248413N). P.S. is holder of a fundamental clinical research mandate by FWO-Vlaanderen (Flemish fund for scientific research).
Trial registration number: Not applicable},
  author       = {Neupane, Jitesh and Vandewoestyne, Mado and Heindryckx, Bj{\"o}rn and Ghimire, Sabitri and Lu, Yuechao and Qian, Chen and Lierman, Sylvie and Van Coster, Rudy and Gerris, Jan and Deroo, Tom and Deforce, Dieter and De Sutter, Petra},
  issn         = {0268-1161},
  journal      = {HUMAN REPRODUCTION},
  keyword      = {preimplantation embryos,preimplantation genetic diagnosis,mtDNA heteroplasmy,mitochondrial DNA,mtDNA disorders,MUTATION LOAD,POLAR BODIES,DNA,MTDNA,SEGREGATION,OOCYTES,PGD,TRANSMISSION,INHERITANCE,BLASTOMERES},
  language     = {eng},
  number       = {4},
  pages        = {852--859},
  title        = {A systematic analysis of the suitability of preimplantation genetic diagnosis for mitochondrial diseases in a heteroplasmic mitochondrial mouse model},
  url          = {http://dx.doi.org/10.1093/humrep/deu016},
  volume       = {29},
  year         = {2014},
}

Chicago
Neupane, Jitesh, Mado Vandewoestyne, Björn Heindryckx, Sabitri Ghimire, Yuechao Lu, Chen Qian, Sylvie Lierman, et al. 2014. “A Systematic Analysis of the Suitability of Preimplantation Genetic Diagnosis for Mitochondrial Diseases in a Heteroplasmic Mitochondrial Mouse Model.” Human Reproduction 29 (4): 852–859.
APA
Neupane, J., Vandewoestyne, M., Heindryckx, B., Ghimire, S., Lu, Y., Qian, C., Lierman, S., et al. (2014). A systematic analysis of the suitability of preimplantation genetic diagnosis for mitochondrial diseases in a heteroplasmic mitochondrial mouse model. HUMAN REPRODUCTION, 29(4), 852–859.
Vancouver
1.
Neupane J, Vandewoestyne M, Heindryckx B, Ghimire S, Lu Y, Qian C, et al. A systematic analysis of the suitability of preimplantation genetic diagnosis for mitochondrial diseases in a heteroplasmic mitochondrial mouse model. HUMAN REPRODUCTION. 2014;29(4):852–9.
MLA
Neupane, Jitesh, Mado Vandewoestyne, Björn Heindryckx, et al. “A Systematic Analysis of the Suitability of Preimplantation Genetic Diagnosis for Mitochondrial Diseases in a Heteroplasmic Mitochondrial Mouse Model.” HUMAN REPRODUCTION 29.4 (2014): 852–859. Print.