Assesment of correlation between mitochondrial DNA (mtDNA) copy number and embryo quality.

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Mitochondrial DNA copy number assessed using Next
Generation Sequencing (NGS) as a selection criteria for viable
embryo transfer.
Krzysztof Łukaszuk1,2,3, Sebastian Pukszta 1, Waldemar Kuczyński4,5, Celina Cybulska1, Joanna Liss1
1 INVICTA, Fertility and Reproductive Centre, Gdansk, Poland; 2 INVICTA, Fertility and Reproductive Centre, Warsaw, Poland ; 3 Department of Nursing, Medical University, Gdansk, Poland
4 Centre for Reproductive Medicine KRIOBANK, 15-879 Białystok, Poland; 5 Department of Gynecology and Oncological Gynecology, Medical University, 15-276 Białystok, Poland
Study question
Assesment of correlation between mitochondrial DNA (mtDNA) copy number and embryo quality.

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Summary answer
Elevated mitochondrial DNA copy number is observed in aneuploid embryos, thus indicating that this parameter could become a
viable additional tool for prioritisation of embryos for transfer
What is known already
The number of mtDNA copies can affect all vital processes which take place in cells but mostly the specialized ones requiring most
of the energy for their specialized functions. This could also influence functioning of the gametes and disturb reproductive
possibilities. It known that number and activity of mitochondria correlates with embryo quality but the exact relationship has not
been yet established.

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Study design, size, duration
Retrospective analysis of the amounts of mtDNA in 606 embryos biopsied either on day 3 or day 5 of development. The study was
performed between August 2013 and November 2014 at INVICTA Fertility Centre, Poland.
Participants/materials, setting, methods
Samples analysed during preliminary validation were derived from cytogenetically characterised 173 human cases and their
embryos were previously confirmed to be aneuploid during routine preimplantation genetic screening (PGS) using next generation
sequencing. MtDNA relative amount was assessed as number of reads for mtDNA / number of reads for genome DNA ratio x 1000.
Data are presented as median and quartiles.

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www.invictagenetics.com Table.1 The difference of the relative mtDNA amount
of analyzed parameters.
All analyzed embryos
3 day 5 day
Ploidy status Normal Abnormal
0.004
Normal Abnormal
0.05N 89 204 110 171
mtDNA relative amount 4.6 (2.8-7.2) 5.9 (3.7-8.9) 0.91 (0.6-1.7) 1.1 (0.7-2.1)
Women age status < 37 years >=37 years
0.14
< 37 years >=37 years
0.27N 151 142 136 145
mtDNA relative amount 5.1 (3.2-8.0) 5.7 (3.8-8.6) 1.04 (0.6-1.7) 1.04 (0.7-2.1)
Embryo quality Good Poor
0.09
Good Poor
0.86N 261 32 220 61
mtDNA relative amount 5.7 (3.5-8.6) 4.8 (2.4-6.9) 1.06 (0.6-1.7) 1.02 (0.6-2.1)
Sex
Embryos with
chr. Y
Embryos
without chr. Y
0.16
Embryos with
chr. Y
Embryos
without chr. Y
0.52
N 131 162 126 155
mtDNA relative amount 5.5 (3.8-8.5) 5.0 (3.1-7.3) 0.92 (0.6-1.6) 1.06 (0.6-1.8)
N 39 50 51 59
mtDNA relative amount 4.8 (3.6-7.3) 4.1 (2.2-7.2) 0.77 (0.6-1.4) 1.1 (0.6-1.8)
Transferred embryos
3 day 5 day
Implantation status Implanted Non implanted
0.18
Implanted Non implanted
0.005N 38 24 28 38
mtDNA relative amount
5.0 (3.7-7.8) 4.1 (3.3-4.6) 1.53 (1-2.2) 0.72 (0.5-1.3)

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Fig 1. Comparison of relative mtDNA copy number between
embryos of different ploidy and implantation status.

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Limitations, reasons for caution
A future prospective randomized study could potentially
support the current findings. DNA after WGA is amplified mostly
in two regions – from 65 to 500 (HRV2) and from 9950 to 10270
(Fig.1. This allows us to analyze the relative amounts of
mitochondrial DNA but does not provide sufficient reliability for
more detailed sequence analysis.
Fig 2. Percentage coverage for mitochondrial sequence after next
generation sequencing performed on DNA from single cell after
WGA. .
Wider implications of the
findings:
MtDNA amount has a potential to became an additional
marker improving embryo selection and prioritization for
transfer.

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INVICTA Genetic Laboratory
ul. Trzy Lipy 3, 80-172 Gdansk, Poland,
T: +48 58 58 58 804, E: pgd@invicta.pl;
W: www.invictagenetics.com
About INVICTA Genetic Laboratory
INVICTA is an experienced genetics laboratory (since 2000)
offering wide range Preimplantation Genetic Diagnosis testing
using state of art Next Generation Sequencing (NGS) techniques.
Main results and the role of chance
Our results showed a significant difference in mtDNA amount depending on the stage at embryo’s development. Results from day 3 biopsy
showed much higher mtDNA to genome DNA ratio than those from day 5 (4.47 vs.0.91; p<0.001).
We found higher relative mtDNA amounts in aneuploid vs. normal blastomeres (5.9 vs. 4.6; p=0.004; respectively) and trophectoderms
(1.1 vs. 0.91; p=0.05; respectively). Results from both biopsy materials did not find any correlation between mtDNA copy number and
embryo quality, embryo’s sex or patient’s age. However, we observed a significant difference in mtDNA amount between implanted and
not implanted 5 day embryos (1.53 vs 0.72; p=0.005, respectively).