The average relative quantities of mtDNA observed in association with female age at the cleavage stage.

<p>The mtDNA values (2-^{Delta Delta Ct}) were obtained during real-time PCR analysis. All examined blastomeres were characterised as being chromosomally normal.</p><p>The average relative quantities of mtDNA observed in association with female age at the cleavage stage.</p

Blastocyst mtDNA quantity threshold in relation to clinical outcome.

<p><b>a)</b> The mtDNA quantity viability threshold for euploid blastocysts, established via retrospective analysis of TE biopsies from transferred embryos with known outcomes. All blastocysts producing viable pregnancies contained mtDNA quantities below the 0.003 value (red line) whereas mtDNA quantities above this value were associated with failure to achieve an ongoing clinical pregnancy. <b>b)</b> Results of the prospective blinded study. The mtDNA threshold used was the same as that established in the retrospective study (4a). Validity was confirmed, since all blastocysts producing viable pregnancies contained mtDNA quantities below the cut-off (red line) and no blastocysts with mtDNA quantities above this value achieved an ongoing clinical pregnancy. <b>c)</b> NGS analysis of the mtDNA level in 23 euploid TE samples. The corresponding embryos were transferred during SET cycles, and clinical outcomes were known for 21 of them. As with the real-time PCR experiments, mtDNA levels were lower in the seven implanting embryos (note- the y-axis scale is different for NGS analyses and consequently cut-off values differ).</p

The average relative quantities of mtDNA observed in association to female age and blastocyst chromosome status.

<p>The mtDNA values (2-^{Delta Delta Ct}) were obtained during real-time PCR analysis.</p><p>The average relative quantities of mtDNA observed in association to female age and blastocyst chromosome status.</p

mtDNA quantities and clinical outcomes of 23 TE samples assessed via real-time PCR and NGS.

<p>*The threshold for considering a sample to have elevated mtDNA levels, incompatible with implantation, was 0.003 for real-time PCR and 0.07 for NGS.</p><p>mtDNA quantities and clinical outcomes of 23 TE samples assessed via real-time PCR and NGS.</p

The relationship between mtDNA quantity, female age and embryo chromosome constitution.

<p><b>a)</b> Data obtained during quantitative real-time PCR analysis of TE samples removed from 302 blastocysts demonstrated a statistically significant increase (P = 0.003) in the level of mtDNA in relation to advancing female age. This phenomenon was evident for both euploid and aneuploid blastocysts. <b>b)</b> Real-time PCR analysis of 39 blastomeres showed that cleavage stage embryos from reproductively younger women contained significantly (P = 0.01) higher mtDNA levels, compared to those generated by reproductively older women. <b>c)</b> Real-time PCR analysis of TE samples also demonstrated that aneuploid blastocysts (n = 99) contained significantly (P = 0.025) larger quantities of mtDNA at all ages, compared to those that were euploid (n = 203). Statistical analysis of mtDNA values took place with the use of unpaired two-tailed t-tests.</p

The mtDNA content of chromosomally normal blastocysts in relation to clinical outcome.

<p>On average, chromosomally normal blastocysts capable of establishing a clinical pregnancy contained significantly (P = 0.007) lower levels of mtDNA compared to chromosomally normal blastocysts that failed to do so.</p

mtDNA quantification via NGS analysis of chromosomally normal and abnormal blastocysts.

<p>NGS analysis of TE samples biopsied from 38 embryos showed a statistically significant increase (P = 0.006) in the mtDNA levels occurring in the presence of chromosome errors.</p

Intensity and detected probe concordance comparisons between low and higher 1 ng inputs.

<p>Raw signal intensity correlations between (A) 50 pg (x-axis) and 1 ng (y-axis) UHR total RNA; (B) 10 pg (x-axis) and 1 ng (y-axis) UHR total RNA; (C) single HeLa cell (x-axis) and 1 ng (y-axis) HeLa total RNA. The overlapping sets of detected probes between the low and higher inputs are shown for both the RNA equivalent (D, E) and single cell (F) inputs. All probe values shown are at a threshold of p<0.01.</p

Raw signal intensity correlations between replicates of low input RNAs and whole cells.

<p>(A) 50 pg UHR and BR total RNA and (B) single HeLa and brain tumor (BT) cells; 50 cell tumorsphere (TS) and adherent cells (AC). Pair-wise scatterplots of at least two replicates for each input type are shown for all 29 K probes across the full range of raw signal intensities. Correlations are the square of Pearson's correlation coefficient.</p

Pre-amplification scheme.

<p>(1) First strand cDNA synthesis is primed with tagged oligo-dT and random 9-mer primers. The tagged oligo-dT primer contains a VN anchor followed by a T-30 stretch with a 5′ PCR tag. The tagged random 9-mer consists of a 9-mer followed by the identical 5′ PCR tag. (2) Upon reaching the 5′ terminus of the mRNAs, the reverse transcriptase, via its terminal transferase activity, adds a few nucleotides (predominantly deoxycytidine) to the 3′ end of the newly synthesized cDNAs. (3) The template-switch primer, which consists of the same 5′ PCR tag as well as a 3′ riboguanine stretch, anneals via GC complimentary base-pairing to the 3′ end of the cDNAs, thereby serving as a new template for the reverse transcriptase. (4) After cDNA synthesis, both ends of the cDNAs now contain the identical PCR tag, allowing exponential amplification of the entire cDNA population through single primer PCR (5).</p