Generation of a quantitative mitochondrial index in granulosa cells to assess good-embryo quality in reproductive medicine
Objective: To design, standardize, and verify a quantitative method based on mtDNA content in single-oocyte cumulus cells (CCs) samples to search for a correlation between mtDNA content and embryo quality in patients undergoing assisted reproductive techniques. Design: Cohort study. Materials and M...
| Autor principal: | |
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| Otros Autores: | |
| Formato: | Tesis de maestría acceptedVersion |
| Lenguaje: | Inglés |
| Publicado: |
Facultad de Farmacia y Bioquímica
2016
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| Materias: | |
| Acceso en línea: | http://repositoriouba.sisbi.uba.ar/gsdl/cgi-bin/library.cgi?a=d&c=afamaster&cl=CL1&d=HWA_2812 http://repositoriouba.sisbi.uba.ar/gsdl/collect/afamaster/index/assoc/HWA_2812.dir/2812.PDF |
| Aporte de: |
| id |
I28-R145-HWA_2812 |
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| record_format |
dspace |
| institution |
Universidad de Buenos Aires |
| institution_str |
I-28 |
| repository_str |
R-145 |
| collection |
Repositorio Digital de la Universidad de Buenos Aires (UBA) |
| language |
Inglés |
| orig_language_str_mv |
eng |
| topic |
Mitocondria Índice mitocondrial Células de la granulosa Cuantitativo Medicina reproductiva Calidad embrionaria Mitochondria Mitochondrial index Granulosa cell Quantitative reproductive medicine Embryo Quality Ciencias de la vida |
| spellingShingle |
Mitocondria Índice mitocondrial Células de la granulosa Cuantitativo Medicina reproductiva Calidad embrionaria Mitochondria Mitochondrial index Granulosa cell Quantitative reproductive medicine Embryo Quality Ciencias de la vida López Luna, Almena Montserrat Generation of a quantitative mitochondrial index in granulosa cells to assess good-embryo quality in reproductive medicine |
| topic_facet |
Mitocondria Índice mitocondrial Células de la granulosa Cuantitativo Medicina reproductiva Calidad embrionaria Mitochondria Mitochondrial index Granulosa cell Quantitative reproductive medicine Embryo Quality Ciencias de la vida |
| description |
Objective: To design, standardize, and verify a quantitative method based on mtDNA content in single-oocyte cumulus cells (CCs) samples to search for a correlation between mtDNA content and embryo quality in patients undergoing assisted reproductive techniques. Design: Cohort study. Materials and Methods: Participating patients signed an informed consent; each patient was submitted to a standard ovarian stimulation protocol for IVF and samples of CCs were taken at the time of egg collection. To assess embryo quality a mitochondrial DNA (mtDNA) quantitative analysis in CCs was designed using onestep qPCR. A portion of the D-loop mitochondrial segment was amplified with respect to the IL6 nuclear gene. Identity of the amplicons was confirmed by direct cloning (CloneJet) and automated sequencing (BigDye). A protocol based on phaseseparation was optimized for total DNA and RNA extraction from TRIzol® samples of CCs. The extraction protocol was modified from Thermo Fisher to allow for suitable concentrations and purities of nucleic acids for samples of CCs bearing a small cell number (about 20,000 cells per egg). Profiles of mtDNA content were analyzed and a mitochondrial index was created that reflected mitochondrial content per cohort. A higher mitochondrial index reflects higher mitochondrial content. All experimental samples (40 analyzed CCs) were analyzed using this index and different correlations between established parameters of embryo-quality and mitochondrial content were explored. Results: The extraction of both RNA and DNA rendered suitable concentrations and purities of nucleic acids for downstream analysis. We found an observational relationship between embryo-quality and mitochondrial content in CCs. CCs from embryos that were transferred possessed higher mitochondrial indexes compared to those belonging to embryos that were fertilized but not transferred and this difference is even more pronounced with CCs coming from oocytes that did not fertilize. \nAdditionally, CCs from embryos that followed through day 3 of development after fertilization, higher mitochondrial indexes than those CCs coming from embryos that only followed through day 2, were observed. Similarly, this difference is even more pronounced with CCs coming from embryos that lasted only until day 1. Based on these results a cut-off value of 0.2 in mitochondrial index was suggested for embryo assessment. Conclusion: We generated a quantitative method based on real-time PCR to measure differences in mtDNA content in CCs samples, with the refinement of a method for nucleic acids extraction based on phase separation of TRIzol® samples and we created a mitochondrial index that seems to reflected embryo quality. We propose that CCs holding higher mitochondrial content correspond to those oocytes with greater quality for embryo transfer. Above all, data support the proposal and further exploration of mtDNA content in CCs as a predictor of embryo quality. |
| author2 |
Álvarez, Silvia |
| author_facet |
Álvarez, Silvia López Luna, Almena Montserrat |
| format |
Tesis de maestría Tesis de maestría acceptedVersion |
| author |
López Luna, Almena Montserrat |
| author_sort |
López Luna, Almena Montserrat |
| title |
Generation of a quantitative mitochondrial index in granulosa cells to assess good-embryo quality in reproductive medicine |
| title_short |
Generation of a quantitative mitochondrial index in granulosa cells to assess good-embryo quality in reproductive medicine |
| title_full |
Generation of a quantitative mitochondrial index in granulosa cells to assess good-embryo quality in reproductive medicine |
| title_fullStr |
Generation of a quantitative mitochondrial index in granulosa cells to assess good-embryo quality in reproductive medicine |
| title_full_unstemmed |
Generation of a quantitative mitochondrial index in granulosa cells to assess good-embryo quality in reproductive medicine |
| title_sort |
generation of a quantitative mitochondrial index in granulosa cells to assess good-embryo quality in reproductive medicine |
| publisher |
Facultad de Farmacia y Bioquímica |
| publishDate |
2016 |
| url |
http://repositoriouba.sisbi.uba.ar/gsdl/cgi-bin/library.cgi?a=d&c=afamaster&cl=CL1&d=HWA_2812 http://repositoriouba.sisbi.uba.ar/gsdl/collect/afamaster/index/assoc/HWA_2812.dir/2812.PDF |
| work_keys_str_mv |
AT lopezlunaalmenamontserrat generationofaquantitativemitochondrialindexingranulosacellstoassessgoodembryoqualityinreproductivemedicine AT lopezlunaalmenamontserrat generaciondeunindicemitocondrialcuantitativoencelulasdelagranulosaparaevaluarbuenacalidadembrionariaenmedicinareproductiva |
| _version_ |
1766017515677286400 |
| spelling |
I28-R145-HWA_28122022-02-16 Objective: To design, standardize, and verify a quantitative method based on mtDNA content in single-oocyte cumulus cells (CCs) samples to search for a correlation between mtDNA content and embryo quality in patients undergoing assisted reproductive techniques. Design: Cohort study. Materials and Methods: Participating patients signed an informed consent; each patient was submitted to a standard ovarian stimulation protocol for IVF and samples of CCs were taken at the time of egg collection. To assess embryo quality a mitochondrial DNA (mtDNA) quantitative analysis in CCs was designed using onestep qPCR. A portion of the D-loop mitochondrial segment was amplified with respect to the IL6 nuclear gene. Identity of the amplicons was confirmed by direct cloning (CloneJet) and automated sequencing (BigDye). A protocol based on phaseseparation was optimized for total DNA and RNA extraction from TRIzol® samples of CCs. The extraction protocol was modified from Thermo Fisher to allow for suitable concentrations and purities of nucleic acids for samples of CCs bearing a small cell number (about 20,000 cells per egg). Profiles of mtDNA content were analyzed and a mitochondrial index was created that reflected mitochondrial content per cohort. A higher mitochondrial index reflects higher mitochondrial content. All experimental samples (40 analyzed CCs) were analyzed using this index and different correlations between established parameters of embryo-quality and mitochondrial content were explored. Results: The extraction of both RNA and DNA rendered suitable concentrations and purities of nucleic acids for downstream analysis. We found an observational relationship between embryo-quality and mitochondrial content in CCs. CCs from embryos that were transferred possessed higher mitochondrial indexes compared to those belonging to embryos that were fertilized but not transferred and this difference is even more pronounced with CCs coming from oocytes that did not fertilize. \nAdditionally, CCs from embryos that followed through day 3 of development after fertilization, higher mitochondrial indexes than those CCs coming from embryos that only followed through day 2, were observed. Similarly, this difference is even more pronounced with CCs coming from embryos that lasted only until day 1. Based on these results a cut-off value of 0.2 in mitochondrial index was suggested for embryo assessment. Conclusion: We generated a quantitative method based on real-time PCR to measure differences in mtDNA content in CCs samples, with the refinement of a method for nucleic acids extraction based on phase separation of TRIzol® samples and we created a mitochondrial index that seems to reflected embryo quality. We propose that CCs holding higher mitochondrial content correspond to those oocytes with greater quality for embryo transfer. Above all, data support the proposal and further exploration of mtDNA content in CCs as a predictor of embryo quality. Fil: López Luna, Almena Montserrat. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Buenos Aires, Argentina Álvarez, Silvia Borner, Christoph Facultad de Farmacia y Bioquímica López, Esther Patiño, Bayghen López Luna, Almena Montserrat 2016-11-23 application/pdf Damiano, Alicia Lorenzetti, Mario Mertelsmann, Roland Mitocondria Índice mitocondrial Células de la granulosa Cuantitativo Medicina reproductiva Calidad embrionaria Mitochondria Mitochondrial index Granulosa cell Quantitative reproductive medicine Embryo Quality eng Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica info:eu-repo/semantics/openAccess http://creativecommons.org/licenses/by-nc-nd/2.5/ar/ Ciencias de la vida Generation of a quantitative mitochondrial index in granulosa cells to assess good-embryo quality in reproductive medicine Generación de un índice mitocondrial cuantitativo en células de la granulosa para evaluar buena calidad embrionaria en medicina reproductiva info:eu-repo/semantics/masterThesis info:ar-repo/semantics/tesis de maestría info:eu-repo/semantics/acceptedVersion http://repositoriouba.sisbi.uba.ar/gsdl/cgi-bin/library.cgi?a=d&c=afamaster&cl=CL1&d=HWA_2812 http://repositoriouba.sisbi.uba.ar/gsdl/collect/afamaster/index/assoc/HWA_2812.dir/2812.PDF |