Download mtDNA quantity age and aneuploidy

Objectives
• Understand relationship between mtDNA content and
Female age
Embryo ploidy
Embryo viability
• Consider the technical requirements for mtDNA measurement
• Gain an insight into the current evidence for mtDNA as a
biomarker of embryo viability
Questions:
• Do you think mtDNA might have value as a biomarker of embryo
viability?
A) Yes, quite likely
B) No, highly unlikely
C) Not sure
• Do you think amount of mtDNA can be accurately measured?
A) Yes, measurements should be straightforward
B) Yes, but quantification is challenging
C) mtDNA can not be accurately measured
D) Not sure
Background - Mitochondria
• Involved in vital cellular processes:
• The main cellular powerhouses
• Cells contain from 100 to >150,000
• Each mitochondrion contains one or more copies of its
own genome (mtDNA)
• Little change in mtDNA content until first cellular differentiation
May-Panloup et al., 2005 ; St John et al., 2010
Comprehensive
Examination of mtDNA quantity chromosome
screening
(aCGH or NGS)
Trophectoderm biopsy
Whole genome
amplification
2015
mtDNA quantification
(real-time PCR or
Next Generation Sequencing)
Materials and methods
• Embryo biopsy
• Whole genome amplification
• Quantitative PCR
Amplification of several loci within the mtDNA
Amplification of multi-copy nuclear DNA sequence (Alu)
Normalise mtDNA against nuclear DNA
• Next generation sequencing (not using a low pass PGS strategy)
mtDNA reads counted
nuclear DNA reads counted
Normalise mtDNA against nuclear DNA
mtDNA quantity with age
mtDNA quantity changes with advancing age
Cleavage stage: mtDNA quantity decreases with
advancing female age (P= 0.01)
Result representative of fertilised oocyte mtDNA
Blastocyst stage: mtDNA quantity increases with
advancing female age (P= 0.003)
Result representative of embryonic mtDNA
mtDNA quantity and aneuploidy
mtDNA quantity is associated with embryo ploidy
Elevated mtDNA quantity in aneuploid vs. euploid
blastocysts (P= 0.025)
Analysis of different blastocyst cohort via NGS
Aneuploid blastocysts have higher mtDNA levels vs.
euploid (P= 0.006)
mtDNA quantity relationship with embryo ploidy is independent of female age
mtDNA quantity age and aneuploidy
mtDNA content of blastocyst stage embryos increases with age
mtDNA content is typically higher when embryos are aneuploid
Female age
Blastocyst mtDNA and embryo viability
Retrospective analysis
0,02
mtDNA quantity
92 euploid blastocysts
0,015
28% of euploid
blastocysts that
failed to implant had
elevated mtDNA
Known outcome
0,01
Normal mtDNA levels
0,005
Elevated mtDNA levels
0
Pregnant
Not-pregnant
Fragouli et al., 2015 PLoS Genetics
Conclusion - Biology
Association between mtDNA quantity, age and aneuploidy risk
Implies mitochondrial factor female reproductive aging/aneuploidy?
Elevated mtDNA quantities arise after trophectoderm differentiation
Elevated mtDNA quantities characteristic of embryos under stress ?
Findings consistent with the “quiet embryo” hypothesis ?
Controversy number 1
Several groups have reported confirmatory results
(e.g. Fragouli et al., 2015; Diez-Juan et al., 2015; Spinella et al
2016)
But some have failed to detect a link between mtDNA and viability
(e.g. Victor et al., 2016; Treff et al., 2016)
mtDNA must be normalised against nuclear DNA sequences to
control for differences in the number of cells biopsied
5 cell biopsy specimen
2
2
2
2
2
5 cells = 10 copies of each nuclear gene (autosomal)
Each cell has 5 mitochondria = 25 copies of the mtDNA
Ratio of mtDNA to nuclear is 25:10 = 2.5
mtDNA must be normalised against nuclear DNA sequences to
control for differences in the number of cells biopsied
3 cell biopsy specimen
2
2
2
3 cells = 6 copies of each nuclear gene (autosomal)
Each cell has 5 mitochondria = 15 copies of the mtDNA
Ratio of mtDNA to nuclear is 15:6 = 2.5 (i.e. same testing 5 cells)
Normalisation against a single copy DNA sequence risks error
5 cell biopsy specimen
2
Allele
dropout
affects
~10% of cells
2
1
2
2
Approximately half of
all 5-cell samples will
experience this error.
More than one-third
will see ADO in two
cells increasing
apparent mtDNA
levels by 25%
5 cells = 9 copies of each nuclear gene (autosomal) detected
Each cell has 5 mitochondria = 25 copies of the mtDNA
Ratio of mtDNA to nuclear is 25:9 = 2.8
Measurement of mtDNA - normalization vs. single copy
gene
Samples tested twice – normalization vs. 2 different single copy genes
Are the results for the two genes identical as one would hope?
1,05
1
0,95
0,9
0,85
0,8
No.
0,75
0,7
0,75
0,8
0,85
0,9
0,95
This indicates that results are subject to extreme variability
Single copy genes should not be used for normalization
1
Measurement of mtDNA after normalization using a
Alu or multiple individual genes
Test the same samples twice, once vs. Alu and once with multiple
individual genes averaged together
Are the results identical as one would hope?
2
1
0
4
6
8
10
-1
-2
-3
-4
Yes, very close to identical.
-5
-6
Indicates that accurate quantification from TE biopsy
requires normalization against multi-copy sequences or
multiple distinct genes
12
Controversy number 2
Are we just seeing the effect of morphology
Higher quality trophectoderm has smaller cells, therefore
fewer mitochondria and lower quantities of mtDNA
Extensive comparison of morphology and mtDNA quantity
Morphology only has a small influence on mtDNA levels
TE TE
HighPoor
quality
Few cells
Many
cells
15 mitochondria / 3
4 nuclei
5
= =3.75
Conclusion - Technical
It is essential to normalize mtDNA against nuclear DNA
However, the nuclear DNA sequence must not be a single copy
Morphology has only a small effect on mtDNA levels
Most NGS methods used for PGS do not sequence the mtDNA
adequately to allow reliable quantification
MitoGrade
Study update 1
Prospective clinical evaluation
1,505 embryos tested from 35 clinics
139 (9%) had elevated mtDNA
We have known outcomes for 283 of the embryos:
65.0% led to an ongoing pregnancy and all had ‘normal’ mtDNA
35% failed to implant and one-third of these had elevated mtDNA
Implantation rate for embryos with elevated mtDNA 0% (0/33)
Implantation rate for embryos with normal mtDNA was 73.6%
P<0.0001
MitoGrade
Study update 2
Prospective, blinded, non-selection study (NYU)
200 embryos with MitoGrade and known implantation outcome
Frequency of embryos with elevated mtDNA: 4.5%
Implantation rates
Entire cohort: 143/200 = 71.5%
Embryos with elevated mtDNA: 0/9 = 0%
Embryos with mtDNA in the normal range: 143/191 = 75%
Percentage of non-implanting embryos with elevated mtDNA: 16%
MitoGrade
Study update 2
Proportion of embryos with elevated mtDNA is small at this clinic
Nonetheless the difference in the implantation rate (normal
mtDNA- versus elevated mtDNA) is highly significant (P<0.0001)
Ongoing pregnancy rates
Entire cohort: 143/200 = 61%
Embryos with elevated mtDNA: 0%
Difference in the ongoing pregnancy rate is significant (P=0.0002)
Conclusion - Clinical
What’s relevant for your clinical practice?
Elevated mtDNA quantities characteristic of embryos under stress ?
Potentially valuable for laboratory/media/process optimization?
Embryos with elevated mtDNA extremely rare/absent in some clinics
Threshold of mtDNA content established, above which implantation
of a euploid blastocyst rarely/never occurs
What’s relevant for your clinical practice?
Example: patient 35 year old, blastocyst transfer
Euploid implanting
(40%)
PGS selection:
62% implantation
Euploid not implanting - Unknown reason (25%)
Aneuploid
(35%)
no PGS selection:
40% implantation
What’s relevant for your clinical practice?
Example: patient 35 year old, blastocyst transfer
Euploid implanting
(40%)
Euploid not implanting - Unknown reason
Euploid not implanting - Elevated mtDNA
Aneuploid
(35%)
PGS + MitoGrade selection:
70% implantation
(estimated)
Take-home message
• Some controversy
• When assessing mtDNA quantities, it is essential that accurate methods are used
• Mounting evidence that mtDNA levels are associated with embryo viability
• Randomized trials needed to define the clinical value
Questions:
• Do you think mtDNA might have value as a biomarker of embryo
viability?
A) Yes, quite likely
B) No, highly unlikley
C) Not sure
• Do you think amount of mtDNA can be accurately measured?
A) Yes, measurements should be straightforward
B) Yes, but quantification is challenging
C) mtDNA can not be accurately measured
D) Not sure