Supplementing oocytes with autologous mitochondria enhances fertilization outcomes

Abstract

ObjectiveTo determine if the introduction of autologous mitochondria into developmentally incompetent oocytes enhances developmental outcome following fertilization.DesignUsing a pig fertilization and preimplantation development model, developmentally incompetent oocytes were isolated and supplemented with autologous populations of oocyte mitochondria. The development rates, mitochondrial DNA (mtDNA) copy number, gene expression and DNA methylation patterns up to the blastocyst stage were assessed.Materials and MethodsDevelopmental competence was determined by labeling cumulus-oocyte complexes with brilliant cresyl blue (BCB). Developmentally competent (BCB+) and developmentally incompetent (BCB-) oocytes underwent each of the following treatments: i) in vitro fertilization (IVF); ii) intracytoplasmic sperm injection (ICSI); and iii) autologous oocyte mitochondrial supplementation and ICSI (mICSI). MtDNA copy number and gene expression were determined by real time PCR and RT-PCR, respectively. DNA methylation patterns were determined by bisulfite sequencing. Statistical analysis was performed using ANOVA.ResultsBlastocyst rates for mICSI treated BCB- oocytes were 32.9% ± 16.5 whilst those from IVF equivalents were 7.6% ± 5.8 (P<0.05). Blastocyst rates from mICSI-derived BCB- oocytes and BCB+ oocytes derived through IVF and ICSI were not significantly different. However, blastocysts derived from mICSI treated BCB- oocytes had increased cell numbers (51) compared to BCB+-ICSI derived blastocysts (32). BCB--mICSI-derived embryos increased mtDNA copy number by 444% at the 2-cell stage when compared to non-supplemented oocytes (P<0.01). This represents a 480-fold increase from the metaphase II to the blastocyst stages and that BCB--mICSI-derived blastocysts have similar mtDNA copy numbers to BCB+-derived blastocysts. The pluripotent genes, OCT4, SOX 2, NANOG and REX1, were all present in BCB--mICSI-derived blastocysts at levels comparable or greater than for IVF BCB+-derived blastocysts. Similar outcomes were observed for the trophectodermal marker CDX2 and the early developmental marker H19. DNA methylation of the mitochondrial specific replication factor, POLGA, was similar for all blastocysts analyzed.ConclusionThe results demonstrate that developmentally incompetent pig oocytes (BCB-) can be rescued by supplementation with autologous oocyte mitochondria during ICSI. ObjectiveTo determine if the introduction of autologous mitochondria into developmentally incompetent oocytes enhances developmental outcome following fertilization. To determine if the introduction of autologous mitochondria into developmentally incompetent oocytes enhances developmental outcome following fertilization. DesignUsing a pig fertilization and preimplantation development model, developmentally incompetent oocytes were isolated and supplemented with autologous populations of oocyte mitochondria. The development rates, mitochondrial DNA (mtDNA) copy number, gene expression and DNA methylation patterns up to the blastocyst stage were assessed. Using a pig fertilization and preimplantation development model, developmentally incompetent oocytes were isolated and supplemented with autologous populations of oocyte mitochondria. The development rates, mitochondrial DNA (mtDNA) copy number, gene expression and DNA methylation patterns up to the blastocyst stage were assessed. Materials and MethodsDevelopmental competence was determined by labeling cumulus-oocyte complexes with brilliant cresyl blue (BCB). Developmentally competent (BCB+) and developmentally incompetent (BCB-) oocytes underwent each of the following treatments: i) in vitro fertilization (IVF); ii) intracytoplasmic sperm injection (ICSI); and iii) autologous oocyte mitochondrial supplementation and ICSI (mICSI). MtDNA copy number and gene expression were determined by real time PCR and RT-PCR, respectively. DNA methylation patterns were determined by bisulfite sequencing. Statistical analysis was performed using ANOVA. Developmental competence was determined by labeling cumulus-oocyte complexes with brilliant cresyl blue (BCB). Developmentally competent (BCB+) and developmentally incompetent (BCB-) oocytes underwent each of the following treatments: i) in vitro fertilization (IVF); ii) intracytoplasmic sperm injection (ICSI); and iii) autologous oocyte mitochondrial supplementation and ICSI (mICSI). MtDNA copy number and gene expression were determined by real time PCR and RT-PCR, respectively. DNA methylation patterns were determined by bisulfite sequencing. Statistical analysis was performed using ANOVA. ResultsBlastocyst rates for mICSI treated BCB- oocytes were 32.9% ± 16.5 whilst those from IVF equivalents were 7.6% ± 5.8 (P<0.05). Blastocyst rates from mICSI-derived BCB- oocytes and BCB+ oocytes derived through IVF and ICSI were not significantly different. However, blastocysts derived from mICSI treated BCB- oocytes had increased cell numbers (51) compared to BCB+-ICSI derived blastocysts (32). BCB--mICSI-derived embryos increased mtDNA copy number by 444% at the 2-cell stage when compared to non-supplemented oocytes (P<0.01). This represents a 480-fold increase from the metaphase II to the blastocyst stages and that BCB--mICSI-derived blastocysts have similar mtDNA copy numbers to BCB+-derived blastocysts. The pluripotent genes, OCT4, SOX 2, NANOG and REX1, were all present in BCB--mICSI-derived blastocysts at levels comparable or greater than for IVF BCB+-derived blastocysts. Similar outcomes were observed for the trophectodermal marker CDX2 and the early developmental marker H19. DNA methylation of the mitochondrial specific replication factor, POLGA, was similar for all blastocysts analyzed. Blastocyst rates for mICSI treated BCB- oocytes were 32.9% ± 16.5 whilst those from IVF equivalents were 7.6% ± 5.8 (P<0.05). Blastocyst rates from mICSI-derived BCB- oocytes and BCB+ oocytes derived through IVF and ICSI were not significantly different. However, blastocysts derived from mICSI treated BCB- oocytes had increased cell numbers (51) compared to BCB+-ICSI derived blastocysts (32). BCB--mICSI-derived embryos increased mtDNA copy number by 444% at the 2-cell stage when compared to non-supplemented oocytes (P<0.01). This represents a 480-fold increase from the metaphase II to the blastocyst stages and that BCB--mICSI-derived blastocysts have similar mtDNA copy numbers to BCB+-derived blastocysts. The pluripotent genes, OCT4, SOX 2, NANOG and REX1, were all present in BCB--mICSI-derived blastocysts at levels comparable or greater than for IVF BCB+-derived blastocysts. Similar outcomes were observed for the trophectodermal marker CDX2 and the early developmental marker H19. DNA methylation of the mitochondrial specific replication factor, POLGA, was similar for all blastocysts analyzed. ConclusionThe results demonstrate that developmentally incompetent pig oocytes (BCB-) can be rescued by supplementation with autologous oocyte mitochondria during ICSI. The results demonstrate that developmentally incompetent pig oocytes (BCB-) can be rescued by supplementation with autologous oocyte mitochondria during ICSI.