1

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Human Fertilisation and Embryology Authority

Executive summary3

1. Introduction

9

2. Mitochondrial donation techniques10

3. Effectiveness of MST and PNT

14

4. Safety of MST and PNT

23

5. Clinical considerations

39

6. Recommendations

43

7. Further research46

Annex A: Methodology of the review

55

Annex B: Timeline of considerations

57

Annex C: Evidence reviewed

60

Annex D: Glossary70Scientific review: 2016 update

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The panel continues to see clinical value in maternal spindle transfer (MST) and pronuclear
transfer (PNT) to mitigate or prevent the inheritance of mitochondrial disease. It recommends
that, in specific circumstances, MST and PNT are cautiously adopted in clinical practice where
inheritance of the disease is likely to cause death or serious disease and where there are no
acceptable alternatives. The report describes the reasoning behind this decision and provides
guidance regarding circumstances in which MST and PNT could be considered.
In February 2015 the UK Parliament approved regulations to permit the use of maternal spindle transfer(MST) and pronuclear transfer (PNT), collectively 'mitochondrial donation’, to avoid serious mitochondrial
disease. The regulations, which came into force on 29 October 2015, enable licensed fertility clinics in the
UK to apply to the Human Fertilisation and Embryology Authority (HFEA) for a licence to perform
mitochondrial donation treatments.
Although the regulations make it lawful to use mitochondrial donation in the clinic, the HFEA must be
satisfied that the techniques involved are sufficiently safe and efficacious before any clinic can apply for alicence to offer mitochondrial donation. This report to the HFEA considers the scientific data relevant to anassessment of the safety and efficacy of MST and PNT and makes recommendations on whether either
technique should be introduced into clinical practice. The final decision will rest with the HFEA board.
The process leading to a change in the law in the UK has a long history. The Human Fertilisation andEmbryology (HFE) Act 1990 was amended in 2008 to allow for regulations to be passed to permit
techniques that prevent the transmission of serious mitochondrial disease due to deleterious mutations inmitochondrial DNA (mtDNA), in recognition of research that had taken place over several years. In 2011,
the Government asked the HFEA to examine the safety and efficacy of these techniques and in response
the HFEA established a scientific panel, with broad-ranging scientific and clinical expertise, to examine
the evidence1. Two further reviews were carried out in 20132 and 20143,4. In addition, the HFEA wasasked in January 2012 to carry out a public dialogue5 on the social and ethical impact of making thesetechniques available to patients.
MST and PNT have the potential to avoid transmitting serious mitochondrial disease from mother to child.
In its clinical application, MST involves transferring the nuclear DNA from an oocyte with abnormal
mitochondria and placing it into an oocyte with healthy mitochondria. PNT involves transferring thepronuclei from an embryo that has abnormal mitochondria and placing them into an embryo that has
healthy mitochondria.

1 HFEA 2011 Scientific review of the safety and efficacy of methods to avoid mitochondrial disease through assisted conception.

Accessed at: http://www.hfea.gov.uk/docs/2011-04-18_Mitochondria_review_-_final_report.PDF.
2 Annex VIII: Scientific review of the safety and efficacy of methods to avoid mitochondrial disease through assisted conception:
update. Accessed at: www.hfea.gov.uk/docs/Mito-Annex_VIII-science_review_update.pdf.
3 Third scientific review of the safety and efficacy of methods to avoid mitochondrial disease through assisted conception: 2014
update. Accessed at: http://www.hfea.gov.uk/docs/Third_Mitochondrial_replacement_scientific_review.pdf.
4 Addendum to the 2014 update - Review of the safety and efficacy of polar body transfer to avoid mitochondrial disease.
Accessed at: http://www.hfea.gov.uk/docs/2014-10-07_-_Polar_Body_Transfer_Review_-_Final.PDF.
5 HFEA Public Dialogue: Medical frontiers: debating mitochondria replacement. Accessed at: http://www.hfea.gov.uk/9359.html.

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This fourth scientific review of the safety and efficacy of mitochondrial donation follows a similar structure
to the earlier reviews. As before, the aim is to provide a comprehensive overview of the scientific issues
raised by mitochondrial donation and an assessment of the current state of the research. The panel was
tasked with reviewing the latest evidence of safety and efficacy for the two mitochondrial donation
techniques – MST and PNT, with particular reference as to whether the recommendations outlined in the2014 scientific report have been met. However, whereas the previous reports were commissioned by theGovernment to inform a decision about whether to change the law, the context for this report is different:
Parliament has changed the law and this report has been commissioned by the HFEA as part of aninternal assessment of whether mitochondrial donation is ready for clinical practice.
In deciding to change the law Parliament has always been clear that neither MST nor PNT should be
introduced into clinical practice until they were judged sufficiently safe. Before the first application forlicensing can be received, the outstanding safety and efficacy experiments recommended in the 2014
scientific mitochondria review (see Box 1) need to be considered by the panel and then by the HFEA3.



MST using human oocytes that are then fertilised (not [artificially] activated): It is still important forsome follow-up experiments to be carried out, notably to improve efficiency, if possible, and further
corroborative experiments would be valuable.



Experiments comparing PNT using normally-fertilised human oocytes with normal ICSI fertilised
human oocytes: The method continues to be developed and appears promising. Further work will be
published in the near future and those results will need assessing before they can be incorporated
into recommendations.



PNT in a non-human primate model, with the demonstration that the offspring derived are normal, is
not critical or mandatory.



MST and PNT should both be explored and that, as yet, it did not consider one technique to bepreferable to the other.

Consideration should be given to mtDNA haplogroup matching (see section 3.7.20 of the 2014
report) as a precautionary step in the process of selecting donors.

This fourth review was commissioned in response to recent publications reporting significant progress inaddressing the recommendations above6,7,8. Whilst these publications were the trigger, the review also
6 Hyslop LA, Blakeley P, Craven L, Richardson J, Fogarty NM, Fragouli E, Lamb M, Wamaitha SE, Prathalingam N, Zhang Q,

O’Keefe H. Towards clinical application of pronuclear transfer to prevent mitochondrial DNA disease. Nature. 2016 Jun 8 534:
383-386.
Hyslop LA, Blakeley P, Craven L, Richardson J, Fogarty NM, Fragouli E, Lamb M, Wamaitha SE, Prathalingam N, Zhang Q,
O’Keefe H. Corrigendum: Towards clinical application of pronuclear transfer to prevent mitochondrial DNA disease. Nature. 2016
Jul 27 538, 542.
7 Yamada M, Emmanuele V, Sanchez-Quintero MJ, Sun B, Lallos G, Paull D, Zimmer M, Pagett S, Prosser RW, Sauer MV,
Hirano M. Genetic Drift Can Compromise Mitochondrial Replacement by Nuclear Transfer in Human Oocytes. Cell stem cell.
2016 Jun 2;18(6):749-54.
8 Kang E, Wu J, Gutierrez NM, Koski A, Tippner-Hedges R, Agaronyan K, Platero-Luengo A, Martinez-Redondo P, Ma H, Lee Y,
Hayama T, Van Dyken C, Wang X, Luo S, Ahmed R, Li Y, Ji D, Kayali R, Cinnioglu C, Olson S, Jensen J, Battaglia D, Lee D, Wu
D, Huang T, Wolf DP, Temiakov D, Izpisua Belmonte JC, Amato P, Mitalipov S. Mitochondrial replacement in human oocytes
carrying pathogenic mitochondrial DNA mutations. Nature 2016 DOI: 10.1038/nature20592

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took into account other relevant studies conducted since the previous review, in order to consider whether
the techniques are now ready to be used in clinical practice and, if so, what clinical issues should betaken into account.
This review builds on the findings of the previous three reviews (including the addendum to the third
review, exploring polar body transfer techniques), and is not written as a stand-alone summary of current
scientific knowledge. For full details of areas reviewed, please refer to the previous reports. An updated
timeline highlighting the key developments in the consideration of mitochondrial donation in the UK is at
Annex B.
As noted above, the science relevant to the safety and efficacy of mitochondrial donation has been
considered in detail by this panel a number of times since 2011. This has allowed the direction of travel tobe assessed in addition to the current state of the methodology. On each occasion, the panel has
reviewed evidence from experts directly in the field, both in the UK and abroad, and taken account of
unpublished as well as published data and opinions. The panel’s reports to Government in 2011, 2013,2014, and now this one to the HFEA in 2016, reflect this composite evidence.
In 2016 the panel considered submissions received as a result of the call for evidence and reviewed newliterature in this area, as set out in Annex C. The panel met on five occasions to allow some of those who
had submitted evidence to present their work and take part in a roundtable discussion. These individuals
or groups had been selected because of the direct relevance of their work to the methods being
considered. The panel was therefore able to consult with a number of relevant research groups andadditional experts in order to inform their conclusions on the progress of current research.

The panel agreed that good progress had been made in experiments recommended in previous reviews
and that additional clinical data should also be considered:

MST using human oocytes that are then fertilised (not artificially activated): There has been significant
progress in this area of research from the group of Professor Shoukrat Mitalipov and his collaborators8
demonstrating an increase in the efficiency of these methods using oocytes carrying pathogenic
mtDNA mutations as well as from controls. Carryover of mtDNA was below 1% in MST blastocysts(Kang et al 2016). Whilst Yamada et al. (2016) used artificially activated rather than fertilised oocytes
following MST, they report that the process of oxidative phosphorylation (OXPHOS) was normal indifferentiated cell types from embryonic stem (ES) cells derived from MST embryos, despite using
mtDNA combinations from distinct haplogroups. Similar results were also obtained by Kang et al(2016). Yamada et al. (2016) further corroborated these conclusions by studying ES cells derived fromsomatic cell nuclear transfer (SCNT) blastocysts.

Experiments comparing PNT using normally-fertilised human oocytes with normal ICSI-fertilised
human oocytes: Hyslop et al., (2016) demonstrated that PNT undertaken shortly after in vitro
fertilisation resulted in the generation of embryos that were competent to develop to the blastocyst
stage. Carryover of mtDNA was shown to be less than 2% and detailed characterisation of theseblastocysts indicated no detectable increase in the incidence of aneuploidy or disruption to normal
profiles of gene expression in isolated cells.



The use of PNT to establish a pregnancy for a 30-year old nulligravid woman with unexplained
infertility was published by Zhang et al. (2016a)9. After transfer of five embryos, a triplet pregnancy
resulted that was surgically reduced to twins. The two remaining fetuses survived only to mid-

9 Zhang J, Zhuang G, Zeng Y, Grifo J, Acosta C, Shu Y, Liu H. Pregnancy derived from human zygote pronuclear transfer in apatient who had arrested embryos after IVF. Reproductive BioMedicine Online. 2016a Oct 31;33(4):529-33.

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gestation, probably due to the obstetric complications of the multiple pregnancy, rather than the PNT
itself. Neither fetus had detectable levels of the maternal (karyoplast-derived) mtDNA haplotype.


A recent abstract, entitled “First live birth using human oocytes reconstituted by spindle nuclear
transfer for mitochondrial DNA mutation causing Leigh syndrome” by Zhang et al. was published online
in the programme for the October 2016 American Society for Reproductive Medicine (ASRM) meeting.
The baby is reported to be doing well and was found to have low levels of mutated mtDNA in several
tissues. Some details of the MST methods used are given by the authors in another abstract in thesame programme10. However, the full details of the methods and treatment cycle have not yet been
published.

In previous reviews the panel recommended experiments on mtDNA behaviour in human embryonic stem(ES) cells (and their differentiated derivatives) derived from blastocysts generated by MST and PNT.
Although such experiments do not accurately model the developing fetus, they do permit an analysis of
the mtDNA carried over in the karyoplast in conditions where mtDNA replication occurs. There have been
three studies6,7,8, all of which report that in the majority of ES cell lines the karyoplast-derived mtDNA
haplotype remained at a similarly low level as in the blastocysts from which the cells were derived, evenafter many passages, or was lost altogether.
However, each study also reported exceptions to this, in which the karyoplast-derived mtDNA haplotype
increased in proportion to that of the cytoplast (one out of eight in Yamada et al., 2016; one out of five inHyslop et al., 2016; and three out of 18 in Kang et al. (2016)). This “reversion”11 was also seen in two out
of 12 ES cell lines derived after SCNT by Yamada et al. (2016) and in one out of eight such lines
described in Kang et al. (2016). Whilst the degree of reversion was variable and seemed stochastic whensubclones of the ES cells were analysed, it could approach 100%. Data indicate that reversion may also
occur in cells differentiating from ES cells7,8, suggesting that it is not an issue specific to ES cells
maintained in a pluripotent state.
Moreover, reversion can occur with normal or mutant mtDNA. Therefore, despite the findings of Zhang etal. (2016a and b), the panel could not rule out the possibility of reversion occurring in clinical application ofthese techniques. These experiments (Yamada et al., 2016; Hyslop et al., 2016; Kang et al., 2016), andtheir possible significance, are considered in detail in sections 3 and 4. Ideally, it would be possible to
avoid any chance of reversion, an eventuality which may depend on understanding the underlying cause.
Some possibilities are discussed in section seven.

The panel continues to note that available data do not indicate whether one technique, MST or PNT, ispreferable to the other at this stage and recommends that both should be considered. However, it notesthat PNT is currently more refined within the UK. The panel also reiterates its opinion that polar body
10 Zhang J, Liu H, Luo S, Chavez-Badiola A, Liu Z, Munne S, Konstantinidis M, Wells D, Huang T. First live birth using humanoocytes reconstituted by spindle nuclear transfer for mitochondrial DNA mutation causing Leigh syndrome. Fertility and Sterility.
2016b Sep 1;106(3):e375-6.
Liu H, Lu Z, Luo S, Chavez-Badiola A, Blazek J, Munne S, Huang T, Zhang J. In vitro fertilization and development of humanoocytes reconstituted by spindle nuclear transfer to replace mutated mitochondrial DNA. Fertility and Sterility. 2016 Sep
1;106(3):e21.
11 Different authors have used different terms to describe this phenomenon. In Kang et al. (2016) this is described as “reversion”
because the predominant mitochondrial DNA haplotype found is that of the karyoplast. In Yamada et al. (2016) this phenomenon
is termed “genetic instability” and in Hyslop et al. (2016) “genetic drift”.

Scientific review: 2016 update

Human Fertilisation and Embryology Authoritytransfer (PBT) techniques, discussed in the addendum to the 2014 report4, showed great promise as ameans to minimise mtDNA carryover and avoid mitochondrial disease, but notes that current regulations
and UK legislation do not permit this technique.

The key recommendations are conditional on a number of considerations, including a requirement for
appropriate levels of skill being demonstrated by named practitioners within a named clinic, and relevant
key performance indicators being met, parameters that will be assessed by the HFEA.
From a medical point of view, all novel treatments pose essentially the same question: when is atreatment sufficiently safe to offer to patients? Research cannot answer every question before a newtreatment is offered, nor can it be expected to guarantee safety or efficacy when applied for the first time.
It can only serve to reduce the risk; in this case of a child being born with symptomatic mitochondrial
genetic disease, but with caveats concerning for whom this type of risk reduction strategy might besuitable and highlighting areas that need close attention. Patients must understand and accept thepotential limitations of any proposed treatment, and possible risks, before proceeding. With this in mind,
the panel recommends that it is appropriate to offer mitochondrial donation techniques as clinicalrisk reduction treatment for carefully selected patients.
In coming to this decision, the panel makes some key recommendations for using these techniques inclinical practice.

The panel suggests that MST and PNT should in the first instance be offered to selected patients forwhom preimplantation genetic diagnosis (PGD) would be inappropriate, or unlikely to succeed. Like PGD
undertaken for mtDNA mutations, MST or PNT can be used as a risk reduction strategy12, but initially onlyin those patients in whose germ line there are likely to be high levels of heteroplasmy or homoplasmy forthe abnormal (pathogenic) mtDNA, and who are thus unlikely to have any suitable embryos for transfer.
Pre-treatment assessment would need to take into account the particular mutation involved, theinheritance pattern in the family, the likely clinical manifestations of disease, the efficacy of any previous
treatments such as PGD, and the patient's understanding of the risks and limitations of what is being
offered. If the techniques prove to be safe when used in these patients, including the absence of any
significant reversion to the carried-over mtDNA haplotype, their application could be extended to other
patients.

The panel advises that (i) all patients should be offered prenatal testing if they become pregnant following
MST or PNT treatment, and (ii) centres offering MST or PNT should encourage patients and theiroffspring to take part in long-term follow-up.

The panel continues to recommend that consideration is given to mtDNA haplogroup13 matching as aprecautionary step in the process of selecting donors. As highlighted in the 2014 report, this is a complex
12 Bredenoord AL, Dondorp W, Pennings G, De Die-Smulders CE, De Wert G. PGD to reduce reproductive risk: the case of

mitochondrial DNA disorders. Human reproduction. 2008 Nov 1;23(11):2392-401.
13 A haplogroup is a term used to define a group of similar haplotypes. Mitochondria from separate human lineages can beclassified according to similarities or differences in their DNA sequence into many different haplogroups. The more evolutionary
distant the separation of two maternal lineages, the greater the differences between mitochondrial haplogroups.

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topic, with some potential risks or benefits associated with choosing a specific donor mtDNA
haplogroup/haplotype donor. At present, the panel believes any risks associated with a mtDNA-nuclear
DNA mismatch remain theoretical; the recent studies examining embryonic cells and stem cells generated
from MST- and PNT-derived human embryos reported no evidence of any complications or compromiseof mitochondrial function arising from unmatched mtDNA haplogroups (see section 4.14 – 4.20).
However, the panel recommends that when these techniques are used clinically, the latest evidence
regarding how mtDNA haplotypes affect mitochondrial-nuclear (mito-nuclear) interactions, including
replicative behaviour of mtDNA, should be considered in order to inform the donor selection process.
Such evidence might even indicate the selection of a specific, unmatched donor in any given case.
Whatever decision is made, the panel recommends that haplotype information on the recipient and thedonor is recorded. The panel also noted that in assessing this risk the treating clinician should be mindfulof parallels with potential mito-nuclear mismatch in natural reproduction. Evidence for any effects
associated with particular combinations of mtDNA and specific nuclear alleles in natural reproduction,
perhaps together with any influence of environment, may come from large-scale genome studies linking
DNA sequence with health outcomes14,15.

The panel also highlights some promising areas for continuing research, particularly the exploration of
methods to further reduce or eliminate mtDNA carryover, through refinements of the techniques, andpossible development of new techniques, as discussed above. Furthermore, the panel concludes that it
will be important to decide whether reversion towards karyoplast-derived mtDNA in some ES cell lines
derived from embryos following MST or PNT is clinically relevant and if so, what underlying mechanisms
are responsible. Further research possibilities are discussed in section 7.

14 Horikoshi M, Beaumont RN, Day FR, Warrington NM, Kooijman MN, Fernandez-Tajes J, Feenstra B, van Zuydam NR, Gaulton

KJ, Grarup N, Bradfield JP. Genome-wide associations for birth weight and correlations with adult disease. Nature. 2016 Oct
13;538(7624):248-52.
15 Johnson SC, Gonzalez B, Zhang Q, Milholland B, Zhang Z, Suh Y. Network analysis of mitonuclear GWAS reveals functional
networks and tissue expression profiles of disease-associated genes. Human Genetics. 2016 Oct 4:1-1.

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Mitochondrial malfunction is a significant cause of several serious multi-organ diseases. These
disorders can be due to mutations in nuclear DNA affecting gene products required within
mitochondria, or mutations in DNA carried within the mitochondria themselves. Mitochondrial DNA(mtDNA) encodes 13 of the 90 or so products required exclusively for oxidative phosphorylation(OXPHOS), which generates energy for cells in the form of the energy-storing molecule,adenosine triphosphate (ATP)16. Although relatively rare, the seriousness of these diseases and,in particular, the unusual inheritance pattern of mtDNA mutations has made them a focus forresearch into preimplantation methods to reduce or avoid such diseases in offspring.
The biology of mitochondria is complex and the attendant language is technical in parts. This
report tries to explain the issues in as straightforward a manner as possible and Annex D provides
a glossary with a definition of relevant terms.
The terms of reference for the panel are: ‘to review the latest evidence of safety and efficacy forthe two mitochondrial donation techniques – maternal spindle transfer (MST) and pronuclear
transfer (PNT), with particular reference to whether the recommendations outlined in the 2014
scientific report have been met.’ Accordingly, this review focuses exclusively on the science andthe safety and effectiveness of these techniques; it does not consider the ethical and legal issues
that are raised by such techniques, except when they are directly relevant to proposed research
and/or potential clinical applications.
This report does not contain an in-depth review of preimplantation genetic diagnosis (PGD) toavoid mitochondrial disease because this can be found in the 2014 report (section 2); however,much of the discussion around recommendations and further research draws on a directcomparison between MST/PNT and PGD and their suitability for particular patients.
In February 2015, Parliament approved regulations to permit the use of MST and PNT to avoid
serious mitochondrial disease. The regulations came into force on 29 October 2015. Since thenthe HFEA has designed a system to license and regulate mitochondrial donation. Before a clinic
will be allowed to carry out either of these techniques it will need to follow a two-stage licensing
process: firstly, to apply to the Authority for a general licence for MST and/or PNT, and then to
seek authorisation to undertake the treatment in the case of a particular patient. In making suchan application, clinics will be required to submit information to the Authority about the patient
being treated, including details of the sperm provider and of the mitochondria donor. The clinic will
also be required to have a follow-up scheme in place to monitor any children born as a result of
MST or PNT.

16 Although mitochondria have other functions within cells, such as in lipid metabolism and programmed cell death, these areexclusively encoded by nuclear genes.

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Although prenatal testing and the option of pregnancy termination can be used when the fetus is
at risk of mitochondrial disorder due to mutations in mtDNA, this is not acceptable to somecouples. In addition, because of the peculiarities of inheritance of mtDNA, it is difficult todetermine a safe threshold below which disease is unlikely to manifest; this threshold will differ
according to the specific mutation carried by the patient and, moreover, levels of mutant mtDNA
present in one tissue at one stage may differ from the level elsewhere and at other stages.
Currently, PGD is used as one of the primary methods to avoid the birth of a child who would beborn with, or develop, mitochondrial disease. However, PGD is not suitable for women who carrya high level of abnormal mtDNA in their oocytes, especially when the oocytes are homoplasmic17
for a given mutation18 (estimated to be the case in about 20% of patients at risk), since it isunlikely or impossible that there will be any embryos with a level of abnormal mtDNA low enoughto avoid disease in any resulting children. Therefore, the reliability of the embryo biopsy result,and the level of abnormal mtDNA per embryo, are important factors when assessing the likely
effectiveness and safety of PGD for mtDNA disorders.
In cases where PGD is not appropriate, the transmission of mtDNA disease can be avoided byusing healthy donated oocytes. This method is safe and has strong supporters; however, whilst
this guarantees the disease is not transmitted, it also means that any resultant child will not be
genetically related to the mother.
The novel methods that are the focus of this review, MST and PNT, allow the transmission of bothparents’ nuclear DNA but in effect involve replacing abnormal mitochondria with normal
mitochondria from a healthy donor oocyte. Since the donor oocyte is the intended source of all (orat least a large majority of) mitochondria in the prospective offspring, these techniques have cometo be known as mitochondrial donation techniques, although many still use the term 'mitochondrial
replacement techniques’19.
MST uses micromanipulation techniques to transfer the woman’s nuclear genetic material (thespindle with maternally-derived chromosomes attached) from one oocyte into another from which
the nuclear genetic material has been removed20 (Figure 1). The reconstituted oocyte is thenfertilised with her partner’s sperm (or donor sperm) to trigger embryo development.
PNT uses similar micromanipulation techniques to transfer the nuclear genetic material, but in thiscase both the maternal- and paternal-derived pronuclei from a fertilised oocyte (zygote) aretransferred into an enucleated one-cell embryo (zygote) derived from a fertilised donated egg(Figure 2). MST takes place between metaphase II oocytes (mature eggs) whilst PNT takes place
17 Where all the mitochondria in a cell contain the same mtDNA, which can be either all abnormal or all normal.
18 A woman may have very low levels in her somatic tissues, but homoplasmic oocytes. If the woman herself was homoplasmic, it

is very likely that she would be suffering from mitochondrial disease. The only exception is where this is conditional on otherfactors, such as gene variants in nuclear DNA or environmental effects (e.g. sensitivity to gentamycin-induced deafness).
19 Mitochondrial Replacement Techniques: Ethical, Social, and Policy Considerations, The National Academies Press 2016.
20 This is equivalent to the oocyte being enucleated, and this term is used by some, although the chromosomes are not contained
within a nuclear membrane at this stage.

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between zygotes, after the stage at which the egg has been penetrated by sperm and thematernal and paternal pronuclei have formed, but prior to the breakdown of the pronuclear
membranes, and the first embryonic cell division.
Both techniques are therefore carried out prior to the mingling of maternal and paternal
chromosomes within a one-cell embryo21. With either method, any resulting child would be
genetically unique, inheriting one nuclear genome from each parent, whilst the mitochondria
would largely be derived from the oocyte provided by the donor. These methods could therefore
effectively substitute the mitochondria from the affected woman with mitochondria carrying normal
mtDNA from the oocyte donor. If there were little or no transmission of abnormal mtDNA, thismethod could avoid mitochondrial disease not just in the resulting child, but also in subsequent
generations. Detailed diagrams can be found in Figure 1 and Figure 2.

21 MST occurs pre-fertilisation and PNT occurs post-fertilisation but prior to the breakdown of the pronuclear membranes andsyngamy.

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Figure 1. Maternal spindle transfer (MST)

Human Fertilisation and Embryology Authority

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Figure 2. Pronuclear transfer (PNT)

Bredenoord, A and P. Braude (2010) ‘Ethics of mitochondrial gene replacement: from bench to bedside’ BMJ 341. Images adapted withpermission of the authors.

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In 2014 the panel considered evidence from the research group led by Professor Shoukrat
Mitalipov at the Oregon Health and Science University. The panel was updated on progresswith the macaques produced previously as a result of MST techniques and was informed thatthe four male macaques had then reached, or were approaching, adulthood (five years of age)and that they had shown no signs of abnormalities, but no further follow-up of health outcomes
had been conducted.
The group was seeking to establish the fertility status of the macaques via a breeding
programme and undertaking more focussed studies looking at physiological impact. At thattime, there remained one female macaque who was then 2-3 years old (from a second set ofexperiments), but had not yet reached sexual maturity. In unpublished data, the Oregon group
had further explored details of the methods used for MST with human oocytes, notably to
reduce the rates of abnormal fertilisation and to increase efficiency.

During this 2016 review, the panel considered new evidence from the New York group ofProfessor Dieter Egli, including their recently published work7 and discussion via videoconference; from the Oregon group, via written submissions8 and video conference with Professor
Shoukhrat Mitalipov; and from the New York-based group of Dr John Zhang, via videoconference.
Yamada et al. (2016) used artificially activated rather than fertilised oocytes following MST. They
made use of vitrification (a cryopreservation technique) to avoid having to collect oocytes fromtwo donors at the same time and allow synchronisation of stages. Although either karyoplasts (thespindle plus chromosomes, but inevitably also some cytoplasm, enclosed in plasma membrane),or cytoplasts (the oocyte minus the spindle and chromosomes) could be cryopreserved and used
to obtain embryos after MST, cryopreservation of the former was more efficient. High quality
blastocysts were used to derive a total of eight ES cell lines. Although all steps were reasonably
efficient, the use of artificial activation is not clinically relevant; therefore, these statistics are notdiscussed here. However, it was considered that other aspects of the data obtained werecomparable to experiments where fertilisation has been used (see below).
The percentage of mtDNA carried over with the karyoplast was found to be between 0 and 2.2%,
with an average of 0.2%, remaining constant throughout development to the blastocyst stage. Of
the eight ES cell lines at early passages, four had lost all trace of the karyoplast-associatedmtDNA haplotype. The other four contained between 0.2 and 1.7 %. By passage six,

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no karyoplast-derived mtDNA could be detected in any of the cell lines except one, and this did
not change for at least 30 passages.
However, in the one exceptional cell line in this study, heteroplasmy for the karyoplast-derived
mtDNA increased from 1.3% at derivation to 53.2% at passage 36. Subsequently, bulk culturesfrom this line or subclones derived from it showed seemingly random drift of heteroplasmy byaround passage 40, with some subclones even becoming homoplasmic for either the karyoplastderived or the cytoplast-derived mtDNA haplotype (H1 or L3, respectively). However, the range ofheteroplasmy reduced in the clonal lines around passage 50, and by passage 59, the karyoplast
haplotype returned in bulk culture to the low level (1%) detected in early passages.
Yamada et al. (2016) also made use of ES cell lines derived from blastocysts made by somatic
cell nuclear transfer cloning (SCNT)22,23,24. The mtDNA present within the somatic cell is
introduced along with its nucleus into the enucleated donor oocyte. These SCNT-ES cell lines cantherefore also be used to explore heteroplasmy and its consequences. While ten of the lines
appeared homoplasmic for the donor oocyte mtDNA haplotype, two others showed similar
unstable heteroplasmy as in the one MST-derived ES cell line, with a significant increase in thesomatic cell-derived mtDNA haplotype.
Yamada et al. (2016) obtained differentiated cell types in vitro from both MST-derived and SCNTderived ES cell lines, both to monitor levels of heteroplasmy and to test mitochondrial function.
They found that mtDNA instability could also occur following differentiation, implying that thisphenomenon is not unique to ES cells. See section 4 for a more detailed discussion of the topic ofreversion of mtDNA derived from the karyoplast.
With respect to mitochondrial function, Yamada et al. (2016) analysed respiratory chain enzyme(RCE) activities, oxygen consumption, including coupling efficiency, cell respiratory control ratio,
and spare capacity. The results suggest that the process of OXPHOS was normal in differentiatedcell types from ES cells derived from both MST-derived and SCNT-derived embryos. This was thecase even when using mtDNA combinations from distinct haplogroups.
Yamada et al. (2016) conclude: “the maternal genome is fundamentally compatible with different
mitochondrial genotypes and vertical inheritance is not required for normal mitochondrial function.
Like the paternal genome at fertilisation, the maternal genome can form a novel combination of
nuclear and mitochondrial alleles that is fully functional. This is not unexpected because maternal
nuclear alleles may resegregate with a new mtDNA genotype after inheritance through a son. The
continuous re-combination in human reproduction therefore requires fundamental compatibility of
nuclear and mitochondrial genomes.”
In his published article7 and during the interview, Prof. Egli discussed potential issues ofheteroplasmy. He pointed out that if MST or PNT were used clinically in cases where the patient’s
oocytes were heteroplasmic for mutant and normal mtDNA, and if one or both persisted andamplified, any resulting child could contain the donor (cytoplast)-derived mtDNA haplotype,patient-derived mutant mtDNA and/or patient-derived normal mtDNA, which will be the same
haplotype but differ in the specific mutation. Prof. Egli was concerned not only for the possibility ofreversion to mutant mtDNA, which could lead to mitochondrial disease, but also because of
22 Chung YG, Eum JH, Lee JE, Shim SH, Sepilian V, Hong SW, Lee Y, Treff NR, Choi YH, Kimbrel EA, Dittman RE. Humansomatic cell nuclear transfer using adult cells. Cell Stem Cell. 2014 Jun 5;14(6):777-80.
23 Ma H, Folmes CD, Wu J, Morey R, Mora-Castilla S, Ocampo A, Ma L, Poulton J, Wang X, Ahmed R, Kang E. Metabolic rescue
in pluripotent cells from patients with mtDNA disease. Nature. 2015 Jul 15
24 Noggle S, Fung HL, Gore A, Martinez H, Satriani KC, Prosser R, Oum K, Paull D, Druckenmiller S, Freeby M, Greenberg E.
Human oocytes reprogram somatic cells to a pluripotent state. Nature. 2011 Oct 6;478(7367):70-5.

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concerns that two normal mtDNA genotypes at approximately equal levels could result in alteredmitochondrial function. Such findings have been reported in mice25, although, as was argued in
the panel’s previous reports, their relevance to humans is unclear given that the two mtDNA
haplotypes used were very different and the mice were highly inbred.
Whilst in a distinct genetic context, it is worth noting that a recent study of 12 tissues obtained at
autopsy from each of 152 individuals, ranging in age at death (due to causes other thanmitochondrial disease) from three days to 96 years suggests that tissue- and allele-related
mtDNA heteroplasmy is widespread in human tissue. That this heteroplasmy can be so extensive
is consistent with positive selection for the specific somatic mtDNA mutations, and stochastic or
ratchet effects26,27.
Professor Mitalipov briefed the panel, via video conference, on the status of the macaquescreated previously by MST. The five offspring showed no signs of abnormalities and weredeveloping comparably with controls. Reversion to the mtDNA haplotype associated with thetransferred spindle had not been observed in peripheral tissues (skin, blood and cells from urine).
There appeared to be no effect on fertility of the males: several pregnancies are ongoing. The
single female MST macaque is due to reach sexual maturity in the coming year and will also be
used as part of a breeding program to examine fertility and the transmission of mitochondria tooffspring.
The group of Prof. Mitalipov has further explored details of the methods used for MST with humanoocytes that were then fertilised. Some of this work was provided in the form of a draft manuscript(now published)8 and this, together with additional unpublished work, was discussed with Prof.
Mitalipov in the video conference. These experiments sought to increase the efficiency of themethod, to further test the use of cryopreservation, which could be used to manage the treatmentof the patient independently of the donor (and separate induced ovulation from implantation), andto investigate in vitro reversion to spindle-associated mtDNA. They were able to include a small
number of patients carrying pathogenic mtDNA mutations (causing Leigh Syndrome or MELAS)
and at risk of having affected children to explore how MST might work clinically. Karyoplasts (thespindle plus chromosomes and cytoplasm enclosed in plasma membrane) were introduced byfusion into ‘enucleated’ oocytes (cytoplasts) donated by women with normal mtDNA. The
reconstructed oocytes were then fertilised by ICSI and the resulting embryos allowed to develop
to the blastocyst stage. Some of the latter were used to derive ES cells for further analysis,
notably of karyotypes and to explore the behaviour of mtDNA from both the patient (carried overwith the spindle), and from the donor. Two sets of controls were used for comparisons: embryos
produced after MST between normal donated oocytes, and unmanipulated oocytes (all fertilised
by ICSI).
The cytoplasts that were left over after removing the karyoplasts from the mutant mtDNA carrier
oocytes were analysed to provide a measure of the level of heteroplasmy in each. This wascompared with that seen in biopsies from the patients, in cells biopsied from the blastocysts after
MST, and in ES cell lines were derived from the latter.

25 Sharpley MS, Marciniak C, Eckel-Mahan K, McManus M, Crimi M, Waymire K, Lin CS, Masubuchi S, Friend N, Koike M,

Chalkia D. Heteroplasmy of mouse mtDNA is genetically unstable and results in altered behavior and cognition. Cell. 2012 Oct
12;151(2):333-43.
26 Li M, Schröder R, Ni S, Madea B, Stoneking M. Extensive tissue-related and allele-related mtDNA heteroplasmy suggests
positive selection for somatic mutations. Proceedings of the National Academy of Sciences. 2015 Feb 24;112(8):2491-6.
27 Wallace DC, Chalkia D. Mitochondrial DNA genetics and the heteroplasmy conundrum in evolution and disease. Cold Spring
Harbor perspectives in biology. 2013 Nov 1;5(11):a021220.

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The observations made can be divided into: (1) those that concern the patients and their
management compared with controls, and (2) the outcome of using MST:
1.(a) Estimates of fertility potential (ovarian reserve measured by antral follicle count, Anti
Mullerian Hormone (AMH) and yield of oocytes) in patients suggested they were compromised incomparison with controls; however, as the authors note, the numbers were very low: just four
patients were involved. This contrasts with reports of PGD for mtDNA disorders that show
reasonable numbers of embryos suitable for biopsy can be obtained28,29,30,31.
(b) High fertilisation rates, comparable to controls, were observed using MST oocytes. Abnormal
fertilisation or activation events (e.g. those giving rise to unipronucleate or tripronucleate
embryos) appeared more frequent when using oocytes obtained from the patients compared witheither MST or unmanipulated controls, perhaps related to the lower oocyte yield, and quality; but
again the numbers were low and differences not statistically significant.
(c) Vitrification was used in some cases to allow oocytes from donors and carriers to be obtainedat different times, but this seems not to have been as useful as in the recent studies from the
Newcastle and New York groups6,7.
(d) As anticipated from previous studies31 the levels of heteroplasmy within oocytes from a given
patient correlated better with the clinical picture seen in her previous children than it did with thelevels found in her somatic tissues.
2. (a) All karyoplasts, whether from patients or controls, and cytoplasts, survived the MST
procedures, except for one in the control group. Whilst several embryos produced by MST
showed karyotype abnormalities, the frequency was similar to that seen in unmanipulated
controls. The proportion developing to blastocysts, ~75%, and their quality was also similar
between MST-derived embryos and controls; moreover, this was irrespective of the number ofdifferences (SNPs) distinguishing the mtDNA haplotypes of the two original oocytes used for
MST. The latter could suggest that the potential for such differences to disrupt mito-nuclear
interactions, as proposed to occur in a number of experimental situations involving exchange of
mtDNA haplotypes in inbred animals32, is not realised during human embryo development, at least
until the blastocyst stage.
(b) Out of a total of six blastocysts obtained after MST, using oocytes from the four patients, four
were judged to be of sufficient quality for transfer, one was of low quality and one was aneuploid.
(c) Measurement of levels of karyoplast and cytoplast-derived mtDNA haplotypes in biopsies fromembryos up to blastocyst stages showed that the degree of carryover of the former wasconsistently less than 1%, and this was irrespective of whether the karyoplast came from patientsor controls.
28 Thorburn DR, Wilton L, Stock-Myer S. Healthy baby girl born following pre-implantation Genetic diagnosis for mitochondrial

DNA m. 8993t> g Mutation. In MOLECULAR GENETICS AND METABOLISM 2009 Sep 1 (Vol. 98, No. 1-2, pp. 5-6). ACADEMICPRESS INC ELSEVIER SCIENCE.
29 Monnot S, Gigarel N, Samuels DC, Burlet P, Hesters L, Frydman N, Frydman R, Kerbrat V, Funalot B, Martinovic J, Benachi A.
Segregation of mtDNA throughout human embryofetal development: m. 3243A> G as a model system. Human mutation. 2011
Jan 1;32(1):116-25.
30 Steffann J, Gigarel N, Samuels DC, Monnot S, Borghese R, Hesters L, Frydman N, Burlet P, Frydman R, Benachi A, Rotig A.
Data from artificial models of mitochondrial DNA disorders are not always applicable to humans. Cell Rep. 2014 May 22;7(4):9334.
31 Sallevelt SC, de Die-Smulders CE, Hendrickx AT, Hellebrekers DM, de Coo IF, Alston CL, Knowles C, Taylor RW, McFarland
R, Smeets HJ. De novo mtDNA point mutations are common and have a low recurrence risk. Journal of Medical Genetics. 2016
Jul 22:jmedgenet-2016.
32 Morrow EH, Reinhardt K, Wolff JN, Dowling DK. Risks inherent to mitochondrial replacement. EMBO reports. 2015 May
1;16(5):541-4.

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Human Fertilisation and Embryology Authority(d) The level of the karyoplast-derived mtDNA haplotype remained very low or was absent in the
majority of the ES cell lines, but after prolonged culture, three out of 18, including one from apatient-derived blastocyst, showed a loss of mtDNA of the donor oocyte (cytoplast) haplotype and“reversion” to the mtDNA haplotype associated with the karyoplast. See below for a more detailed
discussion of the topic of reversion of mtDNA derived from the karyoplast.
The group of J. Zhang used MST with human oocytes10. Reconstituted oocytes were fertilised by
ICSI and cultured until blastocyst stages after which a trophectoderm biopsy was performed andtested for mtDNA heteroplasmy carryover and for aneuploidy. Frozen donor oocytes not
employed for MST were used as controls. In contrast to the groups of Mitalipov, Egli and Herbert,
they used an electric pulse to trigger membrane fusion between the karyoplast and cytoplast
rather than a viral fusogen33. Only minimal details of the adapted electrofusion method used were
given in the Zhang et al ASRM abstracts, which report normal rates of development and that 56%
of MST-derived blastocysts were euploid (which would not be expected if there had beenpremature activation), and this was comparable with control blastocysts (53%). These techniques
were applied to oocytes from a patient who carried 24.5% mutated mtDNA at position of 8993
T>G (Leigh syndrome), but whose oocytes probably had a much higher level. (It was reported
that: “She had 4 pregnancy losses and 2 deceased children at age 8 months and 6 years from
Leigh syndrome as confirmed by >95% mutation load.”) Despite a large number of poor quality
oocytes, five MST oocytes were fertilised and four developed to blastocysts, although in this caseonly one was euploid (46 XY) with 5.7% of mutated mtDNA. It should be noted that this appears
to represent a degree of carryover somewhat higher than others have obtained with MST. Thisembryo was transferred to the patient who, it is reported, gave birth to an apparently healthy boy
at 37 weeks gestation. The mutation load in different tissues ranged from undetectable to 9.23%.
Overall these studies indicate that the MST methods worked well, with reasonable success rates,
even with oocytes from women carrying pathogenic mtDNA. Several steps during oocyte retrieval
may have been more inefficient for carriers compared with controls, but this may also reflect theirage distribution and perhaps BMI, and the numbers were too low to reach firm conclusions.
Although there was little evidence of mito-nuclear mismatch in their experiments, Prof. Mitalipov
also suggested that matching donor and recipient mtDNA haplotypes may be desirable.

33 Electrofusion has been used successfully in experiments with mice and other mammals (Tsunoda, Kato and Shioda, 1987;

Kona and Tsunoda, 1988). It had also been tried by other groups working on MST with human oocytes. However, it wasabandoned because it led to poor viability of the resulting embryos. Notably, in experiments by Tachibana et al (2009) on MST
with macaques, it was found to promote activation (and premature completion of meiosis) without fertilisation at much higher
frequencies than Sendai viral fusogen; and similar results were reported by Paull et al (2013) using MST with human oocytes.

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In 2014 the panel was updated on the progress of the Newcastle group’s work, in both written
submissions and presentations at the panel workshop. The panel was informed that the group
had made considerable progress modifying their experiments from those using zygotes thatwere the product of abnormal fertilisation, which they were originally constrained to use, tothose involving normal fertilisation.
Although the products of abnormal fertilisation tend to arrest early in development, irrespectiveof the time of fertilisation, this problem does not typically occur after normal fertilisation, whichallowed the group to establish new procedures and working protocols to improve PNT
efficiency. These and other modifications to the PNT procedures gave reproducibly high rates
of development to the blastocyst stage. A manuscript describing these data was being
prepared by the group for publication.
The group had also proceeded with experiments required to demonstrate the safety andefficacy of these techniques, by determining chromosome make-up, cell numbers, markers ofcell type, and mtDNA carryover. This work had identified some subtle differences in embryo
development that were being investigated, but nothing had been found to raise safetyconcerns. Pyrosequencing analysis had allowed the group to demonstrate that PNT using
normally fertilised human eggs showed very low or absent levels of carryover of mtDNA – lessthan 2%. The group was in the process of deriving ES cells from human embryos created by
PNT.
The Newcastle group was also in the first phase of using MST on human eggs in order tocompare MST and PNT; however, they explained that due to the limited number of human eggs
available for research, it was difficult to carry out comparative MST and PNT experiments inparallel. The group was open-minded as to which technique should be used clinically.
In 2014 the panel concluded that good progress had been made in developing PNT and that
researchers looked likely to make further progress in refining the technique over the short term
to ensure efficacy and in establishing safety (within the constraints of in vitro conditions). As
stated in the 2013 report, the panel still believed that at that time there was insufficientevidence to choose between MST and PNT as a preferred technique.
During this 2016 review, the panel received further information from the Newcastle group, both in
written submission and in a presentation in person. The group has improved their PNT protocol,
by altering the timing of pronuclear transplantation (with respect to the cell cycle stage of both thepronucleus donor and recipient oocytes) and changing the media used in the process. This work,in progress during 2014, has now been published6. By transplanting pronuclei soon after
completion of meiosis (approximately eight hours after insemination by ICSI - early PNT, ePNT),
rather than shortly before the first mitotic division (late PNT, ltPNT), they improved survival ofreconstituted zygotes to 92% (vs 59% for ltPNT). Omission of sucrose used to induce shrinkageof the cytoplasm, and changes to the composition of the medium, also improved blastocyst
survival, but not quite to the levels of controls or autologous PNT. Carryover of mtDNA was
reduced to <2% in 79% of PNT blastocysts; none had carryover >5%. No significant differences ineither aneuploidy rates or profiles of gene expression were found in comparisons between ePNT-

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derived and control blastocysts, both parameters being examined extensively6. This analysis
included single cell RNAseq data, which was clearly able to distinguish cells from the different
lineages present within blastocysts, and to distinguish cells obtained from normal versus
aneuploid embryos. Following modification of the manipulation and embryo culture medium, thegroup found that the PNT manipulations did not have a detrimental effect on blastocyst formationor quality. Although a manuscript posted on the PeerJ preprint server34 has challenged aspects ofthe statistical analysis presented in Hyslop et al (2016), further details provided by the authors of
the latter paper argue that their original analysis and conclusions hold.
The panel concluded that there has been good progress in PNT techniques, which have beenrefined towards optimisation.

In 2014 the panel and consulted experts reflected on whether it was necessary to conduct
studies using oocytes from women affected by mitochondrial disease. Some experts
recommended that as a “gold standard” they would like to see experiments conducted using
oocytes from women affected by mitochondrial disease to see if pathogenic mutations behave
differently from polymorphisms.
The panel discussed the rationale and the extent of this testing, i.e. would it be required forevery type of mutation given that about 300 have been reported to date, which would be
impractical, or for only a few? Moreover, the choice of which to test might depend on thespecific issues to be examined, such as segregation and bottleneck effects, or consequenceson mtDNA-nuclear gene interactions, and whether it is reasonable to extrapolate from one typeof mutation to another.
In 2014 the panel considered evidence from an animal model demonstrating that certain
mtDNA haplotypes may have an advantage on specific nuclear DNA backgrounds. Any study
on pathogenic mutations in the context of MST or PNT would have to control for this, which
may be impossible. Proof-of-principle experiments have been carried out using PNT with micecarrying mutant mtDNA and these rescued respiratory diseases in the derived progeny. There
are now additional mouse models with other types of mtDNA mutation, but while similar PNTexperiments could be carried out with these, they would still be open to the criticism that theymight not reflect the human situation.
The panel raised the considerable ethical and practical issues around such research; whether it
would be reasonable to request women who are keen to achieve an unaffected pregnancy
using MST or PNT to donate eggs to this type of experiment that would not necessarily benefit
their aim, and would require additional exposure to ovarian stimulation. The scientific rationale
would have to be sufficiently strong to ask for this step, especially as some of the women may
34 The panel received an open access, non-peer-reviewed article submission from EH Morrow and FC Ingleby, which claims thatthe Hyslop conclusions are premature until sufficient data are available to carry out statistical modelling. The panel notes that thisdegree of discriminatory power would likely require large numbers of human embryos, which would be neither feasible nor
ethically unproblematic. The panel also received a submission from some authors of Hyslop et at. 2016 rebutting the argumentsof Morrow and Ingleby.

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already have had one or more affected children. Furthermore, as they get older the fertility of
the women is likely to decline and treatment is less likely to succeed.
The panel concluded, in 2014, that this is an area where a recommendation could be revisitedin the transition between the current research on the safety and efficacy of the techniques andany future consideration of how early clinical use might be conducted. It does not consider thisan obligatory step at present. Moreover, and bearing in mind the ethical issues outlined above,
if incorporated prior to early clinical use it would be reasonable to choose only one or a few ofthe most common types of mutation. In addition, clear experimental objectives would need to
be defined, which would depend on the nature of any prevailing questions and data concerning
any potential replicative advantage attributed to the specific mutation in question.
In 2016 the panel considered the evidence from Prof. Mitalipov and his team (as described inKang et al. 2016), reporting experiments using MST with oocytes obtained from four patients whocarried abnormal mtDNA and were at risk of having affected children. This was clearly a useful
exercise for his team, and it revealed some of the complications of conducting IVF procedureswith the patients. However, apart from the need to coordinate oocyte collection from donors aswell as the patients, which can be managed through cryopreservation, these issues would be
similar to those encountered when using PGD to avoid mitochondrial disease. In terms of the useof the MST techniques and results obtained with them, these were no different from theexperiments conducted in parallel with oocytes from normal donors. There is also a report of therecent birth of a child after MST for a patient carrying a mtDNA mutation (8993 T>G) (see 3.15
above); however, insufficient details are currently available, and the numbers involved are too low
to draw any general conclusions.
The panel continues to believe that conducting studies using oocytes from women
affected by mitochondrial disease is not an essential area of research, in advance ofclinical use.

In 2014, following further discussion with researchers, the panel upheld the view that the use of
non-human primate experiments was no longer critical and raised this for discussion at thescientific workshop in 2014. One expert thought that currently successful macaque trials
exploring MST techniques do not invalidate the possibility of mito-nuclear incompatibilities,
despite the use of two genetically distant subpopulations, and that the current studies haveinsufficient statistical power to detect mito-nuclear incompatibilities.
Demonstrating that mito-nuclear interactions are not of practical concern would require a muchlarger sample of macaques. An experimental design that would be statistically significant would
involve >5 genotypes, with replicate observations, resulting in approximately 40 offspring in
total. Trans-generational effects would require breeding and monitoring of subsequent
generations.
Although these reviews are focused on the science, there are ethical concerns about carryingout experiments on animals, especially non-human primates, if these are likely to not be
informative. Therefore, as stated in 2013, given that the most critical species in which to obtain

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results is the human, and because there are differences in the very early embryology between
mammalian species, the panel also concludes that if any additional experiments on MST and
PNT in other animal models reveals differences from humans, it would be not just reassuring,but important if such experiments revealed the underlying reasons, and did not merely state theproblem.
It is therefore the panel’s view that such experiments would be difficult to justify with respect tocost, ethical considerations and the length of time required, especially as any risks wereconsidered to be small. The panel suggested that a more informative experimental approach
would be based on the derivation of embryonic stem cell lines and subsequent differentiation ofthese to examine oxidative phosphorylation, gene expression levels, and other physiologicalparameters in distinct human lineages derived from MST/PNT embryos.
During this 2016 review, the panel did not receive any evidence to indicate that other groups have
had success with PNT in macaques, nor that anyone is attempting such experiments. The ethicalarguments above still stand: it would be a major undertaking to embark on a study of PNT in
macaques, or other non-human primates, when the methods need to be established, when there
are clear differences in the biology of early macaque and human embryos that may prevent this,and when PNT does work with human zygotes, which is of direct relevance. Furthermore, many ofthe questions that these experiments might address, namely issues of mtDNA carryover and its
subsequent behaviour in offspring, can be addressed by studying the macaques derived by MST.
Other work, for example, studying the behaviour of mutant mtDNA after mitochondrial
replacement, is best performed in a non-primate animal model where this has already been
derived, namely the mouse, or in human embryos and cell lines in vitro.
As in previous years, the panel believes that PNT in a non-human primate model, with thedemonstration that the offspring derived are normal, is not required.

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The safety of MST and PNT has been a central concern of each review. The first review
undertaken in 2011 was based on studies published up to March 2011 (outlined in section 4.3 ofthe original report). In 2013, the panel re-examined and commented on the following safetyissues: the carryover of mtDNA from the affected oocyte or zygote; the methods to prevent
premature activation of oocytes or detect abnormally fertilised oocytes; the mito-nuclear
interactions involved and the potential for long-lasting nuclear epigenetic modifications resulting
from manipulation or altered mitochondrial states associated with mitochondrial disease. The
panel revisited these topics in 2014, updating their views on each strand, where applicable35. In
2016 the panel also considered these areas, with an additional focus on considering work using
embryonic stem (ES) cell lines to understand the fate of mtDNA that had been carried over withthe karyoplast.

In previous reports the panel highlighted the potential concern for subsequent generations if afemale child born after the use of these techniques had a proportion of oocytes with asignificant level of heteroplasmy. It suggested that this could be investigated by, for example,
following differentiation protocols reported to generate primordial germ cells from human ES
cells in vitro. Alternatively, it may be sufficient to explore these ‘bottleneck’ issues by looking at
clonal ES cell lines. If it turned out that there was a significant risk that a proportion of oocytes,
and therefore any resulting embryos from a women born after MST or PNT, could beheteroplasmic, then a recommendation might be made for her to make use of PGD to select forembryos homoplasmic for the normal mtDNA variant, or with very low heteroplasmy.
In 2013, the panel concluded that any early segregation of a very low level of mutant mtDNA
was unlikely to be a problem for children born as a result of MST (or PNT). In 2014, the panel
established that no new evidence had been presented in relation to this area. The panel
continued to recommend that, if any uncertainty about the degree of heteroplasmy in oocytes
remains when women born as a result of MST or PNT wish to have children, then either thisshould be examined directly in their unfertilised oocytes collected after ovarian stimulation,
and/or PGD should be carried out on fertilised embryos prior to selecting those with no, or very
low levels of, abnormal mtDNA for transfer. This should minimise the chance of transmission ofabnormal mtDNA to subsequent generations.
Carryover of mtDNA from the affected oocyte or zygote is expected with both MST and PNT
because, during micromanipulation, the spindle or the pronuclei are surrounded by a small
amount of cytoplasm that contains mitochondria. Evidence presented to the panel in 2013, andsince, confirms that carryover after mitochondrial replacement is very low, and refinements in
35 In 2014, the panel discussed the role of reagents used in both techniques and concluded that the MHRA would need to besatisfied about the provenance and clinical safety of all reagents. The panel were satisfied, in 2016, that the clinical safety of all
reagents was being considered, and validated.

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techniques of karyoplast extraction have continued to reduce it for PNT (including ePNT), andMST, such that the levels are <2% in the vast majority of embryos tested6,7,8. Levels of abnormal
mtDNA of 2% would not be expected to cause pathologies; indeed, many individuals probably
exhibit similar levels in somatic tissues without any obvious consequences36,37. Moreover, muchhigher levels of heteroplasmy than 2% are deemed acceptable in PGD for mtDNA mutations.
However, carryover of abnormal mtDNA following MST or PNT may cause problems
subsequently if it is preferentially amplified (perhaps by effects on mtDNA replication) and/or if
there is a marked difference in segregation across tissues, either because of factors specific tothese methods or because of the presence of two different mtDNA haplogroups/haplotypes.
The panel also considered that by extrapolating data on macaques derived by MST, if the childwere female, then while some of her oocytes may have very low or undetectable levels of mutant
mtDNA, it is possible that others have a significantly higher proportion, considerably higher thanher somatic tissues. These levels may still not be sufficient to predispose her children to manifest
disease, but subsequent generations could be affected. Although diagnostic technology may wellhave advanced by then, the use of PGD (on embryos38 from oocytes of female offspring resulting
from MST or PNT, who might be carriers of the mutation in some of their oocytes) would enable
the preferential selection for implantation of embryos whose tested blastomeres or trophoblast
cells had low levels of abnormal mitochondria30. This could reduce the risk of mitochondrial
disease in subsequent generations.

In this 2016 review, the panel considered recent data by Yamada et al. (2016) and the Newcastle
group6 on ES cells obtained from parthenogenetic MST embryos and normally-fertilised PNTembryos, respectively. It also considered the data from the laboratory of Prof. Mitalipov8 that
described MST in human oocytes that were normally fertilised (now published8). All three groups
sought to assess the potential fate of mtDNA that had been carried over with the karyoplast by
deriving human embryonic stem (ES) cell lines. Hyslop et al. (2016) derived five ES cell lines fromePNT blastocysts, having also assessed the levels in the blastocysts from trophectoderm
biopsies.
While all PNT-ES cell lines showed low levels of heteroplasmy at passage 1 (P1), and three of thelines retained this low level after extensive culture, one line (36PNT), derived from a blastocyst
with 4% mtDNA carryover, was unique in showing an upward drift with wide variation in levels ofheteroplasmy between colonies by P12. The group confirmed this by experiments in which
individual colonies from the original line were subcloned and cultured for multiple passages (thefifth hES cell line was not studied beyond passage 2). Hyslop et al. highlighted that the karyoplastand cytoplast donors for the cell line showing this upward drift in karyoplast-derived mtDNA
belonged to the same mtDNA haplogroup, whereas the other three lines studied all involved
36 Greaves LC, Nooteboom M, Elson JL, Tuppen HA, Taylor GA, Commane DM, Arasaradnam RP, Khrapko K, Taylor RW,

Kirkwood TB, Mathers JC. Clonal expansion of early to mid-life mitochondrial DNA point mutations drives mitochondrial
dysfunction during human ageing. PLoS Genet. 2014 Sep 18;10(9):e1004620.
37 Ye K, Lu J, Ma F, Keinan A, Gu Z. Extensive pathogenicity of mitochondrial heteroplasmy in healthy human individuals.
Proceedings of the National Academy of Sciences. 2014 Jul 22;111(29):10654-9.
38 Another possibility would be to test polar bodies for levels of heteroplasmy, however, this is known to be very unreliable – see
Gigarel N, Hesters L, Samuels DC, Monnot S, Burlet P, Kerbrat V, Lamazou F, Benachi A, Frydman R, Feingold J, Rotig A. Poor
correlations in the levels of pathogenic mitochondrial DNA mutations in polar bodies versus oocytes and blastomeres in humans.
The American Journal of Human Genetics. 2011 Apr 8;88(4):494-8.

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combinations of distinct haplogroups. This suggests that there is no simple relationship between
degree of match of mtDNA haplogroups and risk of amplification of carried-over mtDNA.
Yamada et al. (2016) independently assessed the potential for changes in the degree ofheteroplasmy following human mitochondrial donation, using proliferating stem cell lines and invitro differentiation models, in which mtDNA replication is common. They utilised human oocytes
reconstructed by MST that were subsequently activated without fertilisation to yieldparthenogenetic embryos. These embryos, which were capable of forming high quality
blastocysts, had consistently low levels of karyoplast-derived mtDNA carryover (average of 0.2%).
To assess whether this low level heteroplasmy was stable following cell division, stem cell lines
were derived from blastocysts. For all but one of the eight stem cell lines derived, heteroplasmy
levels decreased below the limit of detection from passage 6 and all subsequent passages (morethan 6 months of culture)39. However, in one cell line, levels of karyoplast-derived mtDNA
increased during culture to 53.2% at passage 36. At passage 59, however, levels had reduced to
1.0%. Fluctuation was also observed when affected ES cell lines were differentiated.
Additional experiments in the same study used SCNT to introduce diploid nuclei from humansomatic cells into human oocytes. SCNT also introduces approximately one thousand somatic cell
mitochondria into the oocytes, therefore ES cell lines derived from SCNT-derived blastocysts
were also examined for heteroplasmy. They examined four diploid SCNT ES cell lines, in which
the oocyte had been enucleated prior to fusion with the somatic cell, and eight lines that hadretained the oocyte genomic DNA. Nine of the lines showed very low heteroplasmy, with thesomatic cell mtDNA haplotype ranging from 0-0.6 %.
However, two of the diploid SCNT ES lines that had arisen from the same somatic cells andoocyte donor showed a rapid upward drift of the somatic mtDNA haplotype within 10 passages
and had reached homoplasmy between passage 15 and 25. Clonal analysis confirmed that thedrift was from haplotypes L0 to K1 in both cases. However, a third SCNT ES cell line from thesame original haplotype combination contained no K1 mtDNA and remained homoplasmic for theL0 haplotype from passage 1 to 40. An additional SCNT ES cell line (H1 with L3) also showed
mtDNA instability. When the L0/K1 heteroplasmic ES cells prone to drift were differentiated invitro or in teratomas, there was a similar directional shift to the K1 haplotype, whereas the line
containing H1 and L3 showed drift in either direction. Homoplasmic ES cell lines remained so on
differentiation, as expected.
Further experiments showed no survival or proliferative advantage in culture of homoplasmic K1
ES cells over homoplasmic L0 ES cells, nor were there any significant differences inmitochondrial respiratory chain enzyme (RCE) activity.
Kang et al. (2016) also investigated the stability of low-level heteroplasmy in human embryos
generated by MST followed by normal fertilisation. All MST zygotes and cleaving embryos (n=22)
contained <1% karyoplast-derived mtDNA and similar outcomes were observed in 15 out of 18
ES cell lines derived from MST blastocysts. However, in three of the cell lines (two controls andone from a patient), karyoplast-derived mtDNA predominated, and after extended passaging was
the only detectable mtDNA. They also found that one out of 8 SCNT-derived ES cell lines showeda gradual increase from 19% at passage two to 100% at passage 10.
Thus, three reports of human mitochondrial donation in distinct contexts – including MST followedby normal fertilisation (Kang et al., 2016), parthenogenetic activation (Yamada et al., 2016), andPNT of normally-fertilised oocytes (Hyslop et al., 2016) - report the successful generation of
39 This result is similar to their earlier finding for all of four pluripotent cell lines derived from human spindle transfer blastocysts(Paull et al., 2013).

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embryos exhibiting very low levels of heteroplasmy (carryover typically <1-2%). In the context ofembryonic stem cell culture, in most cases the level of heteroplasmy remained stable or the
karyoplast-derived mtDNA haplotype became undetectable throughout repeated passaging.
However, in a minority of cell lines analysed (in the order of 15-25%) instability of heteroplasmy
was demonstrated, resulting in either a significant increase of, or even complete reversion to, the
mtDNA haplotype found in the karyoplast. This has a clear implication: even though the methods
achieve the desired aims in the preimplantation embryo, were such drift or reversion to occur inthe context of embryonic/fetal development following clinical application of MST or PNT, such thatthe levels of pathogenic mtDNA increased throughout the fetus or in particular tissues, this could
lead to disease in the child born40.
The panel considered several possible explanations for changing levels of heteroplasmy in stem
cells, some of which were suggested in the relevant publications and in discussions with thoseinterviewed by the panel. These relate either to some aspect of the methods used or to propertiesof mtDNA, and they are not mutually exclusive.
(i) Level of carryover: In the report by Hyslop et al. (2016), the one ES cell line derived after
PNT that showed reversion on passaging was that with highest level of carryover (4%), incomparison with <2% for those that did not. However, for the MST-derived ES cell lines (Yamada
et al., 2016; Kang et al. 2016) there was no obvious correlation between reversion and levels ofcarryover; moreover, these tended to be lower, mostly ranging from 0.1-1.0%.
It is also worth noting that none of the embryos and early passage ES cell lines from any of thestudies had carryover levels above 5%, the proposed level above which embryos are theoreticallyat risk of expansion through stochastic/bottleneck effects27,41,42,43. While initial levels of carryover
may not have been a major factor in determining the risk of subsequent reversion in stem cells,
the panel recognises the importance of keeping carryover levels in embryos to a minimum. If
carryover could be eliminated altogether, any risk of reversion would be removed, whether in vitro
or in vivo (see section 7 for possible methods to eliminate or reduce mtDNA carryover).
(ii) Methodology: It is possible that aspects of the methods used in the papers discussed above
could give the karyoplast mtDNA an advantage in some circumstances. For example, the processof SCNT is known on occasion to induce a round of mtDNA replication in the early embryo at atime when this does not normally occur44,45. This may be due to the presence of nuclear-encoded
mtDNA replication factor DNA polymerase  (Pol within the somatic cell, which will be introduced
into the oocyte on fusion. Also, while most proteins present in the oocyte are destroyed during
cleavage stages, some persist, and it is possible that any pol may remain associated with the
mtDNA that originated in the somatic cell, giving this an early advantage when replication begins
in earnest, after establishment of ES cell lines in vitro. However, this mechanism is unlikely to be40

In cases where the patient’s oocytes are heteroplasmic, there is a chance that reversion could involve the normal mtDNA, inwhich case the child would be healthy. Indeed, this was the case in the one patient-derived ES cell line that showed reversion, as
reported in Kang et al., 2016.
41 Johnston IG, Burgstaller JP, Havlicek V, Kolbe T, Rülicke T, Brem G, Poulton J, Jones NS. Stochastic modelling, Bayesianinference, and new in vivo measurements elucidate the debated mtDNA bottleneck mechanism. Elife. 2015 Jun 2;4:e07464.
42 Samuels DC, Wonnapinij P, Chinnery PF. Preventing the transmission of pathogenic mitochondrial DNA mutations: can weachieve long-term benefits from germ-line gene transfer? Human Reproduction. 2013 Mar 1;28(3):554-9.
43 Wilson IJ, Carling PJ, Alston CL, Floros VI, Pyle A, Hudson G, Sallevelt SC, Lamperti C, Carelli V, Bindoff LA, Samuels DC.
Mitochondrial DNA sequence characteristics modulate the size of the genetic bottleneck. Human molecular genetics. 2016 Mar
1;25(5):1031-41.
44 Bowles EJ, Campbell KH, John JC. Nuclear transfer: preservation of a nuclear genome at the expense of its associated mtDNA
genome (s). Current topics in developmental biology. 2007 Dec 31;77:251-90.
45 Lloyd RE, Lee JH, Alberio R, Bowles EJ, Ramalho-Santos J, Campbell KH, John JC. Aberrant nucleo-cytoplasmic cross-talk
results in donor cell mtDNA persistence in cloned embryos. Genetics. 2006 Apr 1;172(4):2515-27.

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responsible for reversion after MST or PNT, because neither of these involve mtDNA in a
replicative state. Yet, the reversion phenomenon occurred in ES cell lines derived after MST, PNTand SCNT. Could there be something else in common?
One possibility might be segregated or partitioned distribution of karyoplast-derived mtDNA after
MST or PNT or SCNT, which could lead to some blastomeres having a level of carryover higher
than others, and perhaps sufficiently high that stochastic/bottleneck effects could operate to skew
the proportion even further during ES cell culture. Relatively few cells of the inner cell mass (ICM)
give rise to ES cells, which could explain why reversion is seen only in a proportion of ES celllines. Single cell analysis of blastocysts should reveal if this is the case. This was carried out by
Hyslop et al. (2016), and it is apparent that a few cells are outliers with up to 12% carryover (seetheir Fig. 3). This is in contrast to evidence reported from PGD of human embryos where
blastomeres within an embryo tend to have very similar levels of heteroplasmy46,47. One obvious
implication of this is that tissues deriving from such blastomeres may have sufficiently high levelsof karyoplast-derived mtDNA that they could be prone to increase to even higher levels, as seen
with one case of PGD47,48 and as predicted by theory49.
The panel discussed the possibility of partitioning followed by segregation after MST and PNT in
its 2013 report, following the studies of Lee et al. (2012)50 who had deliberately created macaqueoocytes that were heteroplasmic (50/50) for two mtDNA variants. These resulted in embryos
exhibiting significant partitioning of the mtDNA between different blastomeres and to some extent
between trophectoderm and ICM. Some of the fetuses, or ES cell lines derived from embryos,
also showed a skewed ratio (in one case about 94% of one of the mtDNA variants was present).
There was no evidence of preferential selection for either mtDNA, suggesting that both variants
function equally well with the resident nuclear DNA, even though the mtDNA sequences of thetwo sub-species of macaque were as different from each other as they are from other primate
species.
Lee et al (2012) also claimed that isolated karyoplasts from macaque oocytes carry “bound”
mitochondria (there is no evidence that they are physically bound, just closely associated) at an
average level of about 0.6% of the numbers within the cytoplasm. Mitochondria interact with thecytoskeleton and this could be involved in subsequent partitioning of the karyoplast-associatedmitochondria. While cytoskeletal inhibitors are used during MST, PNT and SCNT to allow
extraction of the spindle or pronuclei from oocytes or zygotes, which may allow mitochondria tomix freely, the inhibitors are no longer present when the karyoplast containing the spindle orpronuclei (or an intact somatic cell in the case of SCNT), is first washed though a drop of viralfusogen and then fused to the enucleated oocyte or zygote. Perhaps this could allow the
karyoplast cytoplasm and associated mitochondria to remain in a localised region of the zygote,such that they could fail to be partitioned equally amongst blastomeres during cleavage divisions.
It should be possible to follow this using MitoTracker labelling of mitochondria in the karyoplasts,

46 Sallevelt SC, Dreesen JC, Drüsedau M, Spierts S, Coonen E, van Tienen FH, van Golde RJ, de Coo IF, Geraedts JP, de Die-

Smulders CE, Smeets HJ. Preimplantation genetic diagnosis in mitochondrial DNA disorders: challenge and success. Journal of
medical genetics. 2013 Feb 1;50(2):125-32.
47 Treff NR, Campos J, Tao X, Levy B, Ferry KM, Scott RT. Blastocyst preimplantation genetic diagnosis (PGD) of a mitochondrial
DNA disorder. Fertility and sterility. 2012 Nov 30;98(5):1236-40.
48 Mitalipov S, Amato P, Parry S, Falk MJ. Limitations of preimplantation genetic diagnosis for mitochondrial DNA diseases. Cell
reports. 2014 May 22;7(4):935.
49 Chinnery PF, Craven L, Mitalipov S, Stewart JB, Herbert M, Turnbull DM. The challenges of mitochondrial replacement. PLoS
Genet. 2014 Apr 24;10(4):e1004315.
50 Lee HS, Ma H, Juanes RC, Tachibana M, Sparman M, Woodward J, Ramsey C, Xu J, Kang EJ, Amato P, Mair G. Rapid
mitochondrial DNA segregation in primate preimplantation embryos precedes somatic and germline bottleneck. Cell reports. 2012
May 31;1(5):506-15.

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Human Fertilisation and Embryology Authorityand perhaps to prevent it by incubating the reconstituted oocyte or zygote after fusion incytoskeletal inhibitors for a brief period.
(iii) Peculiarities of embryonic stem cells: Among other differences (see box 2 below), ES cells
have a relatively small amount of cytoplasm compared with most somatic cell types and typicallyhave few copies of mtDNA51. They may, therefore, be particularly prone to “bottleneck” effects
where just by chance there is drift in the level of heteroplasmy. This would not, however, be
expected by itself to give a consistent directional drift, as seen in the cases of reversion found by
Yamada et al. (2016), Hyslop et al. (2016), and Kang et al. (2016). But it is noteworthy that the
karyoplast-derived mtDNA haplotype disappeared altogether in some ES cell lines, and in one
line studied by Yamada et al. (2016), which underwent a significant reversion to the karyoplastderived mtDNA haplotype, the effect was completely reversed in bulk culture and subclones also
showed an over-all drift back towards the cytoplast-derived haplotype.
However, even a very small advantage of one mtDNA haplotype over the other could lead to an
additional “ratchet” effect. This could operate within cells, if, for example, one mtDNA haplotype
replicated faster than the other, and/or between cells, if, for example, the advantage involved
energy production, such that cells with slightly higher copy number of the advantageous mtDNA
type within its mitochondria could contribute slightly more of the cells in the next passage, and soon.
Yamada et al. (2016) tested this latter possibility by mixing cells homoplasmic for haplotype L0 or
haplotype K1 at different ratios (in heteroplasmic ES cell lines, drift was consistently in favour of
the K1 haplotype). They found that the proportion of each mtDNA genotype remained stable over
8 passages (6 weeks). This suggests that reversion to the K1 haplotype in cultures ofheteroplasmic ES cells does not involve cell competition52 and that it is more likely to be due to a
replicative advantage of K1 over L0 within cells.
Prof. Mitalipov expressed the view that human embryonic stem cells may not act in the same way
as embryos. Moreover, he informed the panel that he has not observed the same reversion
phenomenon in mouse embryonic stem cells or in those from macaques, suggesting that human
ES cells may be unusual in this regard. Kang et al (2016) report that derivation of ES cell clones
from isolated individual cells from two cell lines exhibiting reversion in whole culture (3243ST-ES1
and NT-ES8) revealed faster growth rates in clones with higher levels of maternal (karyoplastderived) mtDNA. The authors conclude that certain mtDNA haplotypes confer a proliferativeadvantage on ES cells and that this is independent of detectable mitochondrial activity. It remains
unclear how such an effect might occur, given that mitochondria are not very active within ES
cells; moreover, this observation is not consistent with the results of the cell mixing experiments of
Yamada et al. (2016), as described above.
In addition to following mtDNA heteroplasmy in ES cells over many passages, Yamada et al.
(2016) looked at differentiated cell types derived from the same ES cell lines that had shown
dramatic shifts. Differentiation of the two SCNT-derived lines to fibroblasts or cardiomyocytes
resulted in a directional shift to the K1 haplotype, while the MST-derived line showed bothdecrease or increase of the H1 haplotype during differentiation, mirroring what had happened
with further passaging of the undifferentiated ES cells. Cells with homoplasmy remained stable asstem cells and during differentiation in vitro or in vivo as teratomas. The authors conclude:“mtDNA genotype instability was not specific to pluripotent stem cells”.
However, rather than beginning with early passages before any shift was obvious, they used
51 Folmes CD, Ma H, Mitalipov S, Terzic A. Mitochondria in pluripotent stem cells: stemness regulators and disease targets.

Current opinion in genetics & development. 2016 Jun 30;38:1-7.
52 Sancho M, Rodríguez TA. Selecting for fitness in mammalian development. Cell Cycle. 2014 Jan 1;13(1):9-10.

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those that had already shown significant reversion. This is, therefore not an ideal test of what
would happen in somatic cells if embryos derived by MST had been implanted. Nevertheless,
Yamada et al. (2016) also measured the function of mitochondria in the differentiated cells, usinga range of parameters, finding no significant differences between any mtDNA
haplogroup/haplotype. This again suggests that preferential amplification of one haplotype overanother is more likely due to differences in replication and not selection for improved function.

The panel recommends caution when attempting to interpret data from ES and other cells
cultured in vitro in order to make inferences about the risks of mtDNA reversion during
embryonic in development in vivo. ES cells are epigenetically anomalous compared with mostsomatic cells, with pan-genomic loci co-occupied by active (H3K4me3) and silent (H3K27me3)
bivalent marks. Unlike differentiating and differentiated cells, ES cells do not require DNA
methylation53 and ES and somatic cells produce (in the mouse, at least) markedly different
developmental readouts in nuclear transfer, for unknown reasons54. These differences could, ofcourse, influence (or be influenced by) mitochondrial behaviour.
As noted above, ES cells are morphologically atypical, with large nucleus-cytoplasm ratios andthey have an anomalous cell cycle, with a truncated G1 in which Geminin escapes degradation
due to atypical suppression of the E3 ubiquitin ligase, APC55,56,57. The cell cycle is linked tomitochondrial segregation58 and coupled with the unusual morphology of ES cells and theunusual regulation of proteasomal degradation (which is relevant for mitophagy), obviously has
the potential to influence sorting of cytoplasmic factors, including mitochondria, in an unusual
manner.
However, it is not known if these fundamental biological differences between ES and somatic
cells are relevant to mitochondrial behaviour. If ES cells are to be used as a model for whatmight happen in vivo it is necessary to have a closer consideration of the way mitochondria
behave in ES cells compared to somatic cells. The mitochondria of human and mouse ES cells,
unlike their differentiated somatic cell counterparts (e.g. fibroblasts) are immature, with aperinuclear localisation entirely concordant with different mechanisms of mitochondrial
homeostasis.
Moreover, ES cells are derived from a transient compartment in preimplantation embryos thatlasts only a few hours. In addition, no in vitro culture system available today can fully account forexogenous (e.g., circulating) factors (cytokines, growth factors, steroid and peptide hormones,
neurotransmitters, etc.), programmed lineage differentiation, including apoptosis, immune
surveillance, tissue-level reactive oxygen species (ROS) homeostasis, the role of niches, cross53 Li E, Bestor TH, Jaenisch R. Targeted mutation of the DNA methyltransferase gene results in embryonic lethality. Cell. 1992

Jun 12;69(6):915-26.
54 Jackson-Grusby L, Klemm M, Rideout WM 3rd, Yanagimachi R, Jaenisch R Hybrid vigor, fetal overgrowth, and viability of mice
derived by nuclear cloning and tetraploid embryo complementation. Eggan K, Akutsu H, Loring J.Proc Natl Acad Sci U S A. 2001
May 22;98(11):6209-14. Epub 2001 May 1.
55 Yang VS, Carter SA, Hyland SJ, Tachibana-Konwalski K, Laskey RA, Gonzalez MA. Geminin escapes degradation in G1 ofmouse pluripotent cells and mediates the expression of Oct4, Sox2, and Nanog. Current Biology. 2011 Apr 26;21(8):692-9.
56 Gonzalez MA. Curr Biol. 2011 Apr 26;21(8):692-9.; Distinct activities of the anaphase-promoting complex/cyclosome (APC/C)
in mouse embryonic cells.
57 Yang VS, Carter SA, Ng Y, Hyland SJ, Tachibana-Konwalski K, Fisher RA, Sebire NJ, Seckl MJ, Pedersen RA, Laskey RA,
Gonzalez MA. Cell Cycle. 2012 Mar 1;11(5):846-55.
58 Mishra P, Chan DC. Mitochondrial dynamics and inheritance during cell division, development and disease. Nature reviews
Molecular cell biology. 2014 Oct 1;15(10):634-46.

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talk between juxtaposed heterologous cells and tissues, morphogenesis and numerous othersystems-level complexities.
Another complicating factor is that the conditions currently used for establishing and culturing
human ES cells are unlikely to be optimal, because they do not mimic any in vivo environment.
( may mean that ES cells in culture are prone to more stresses than are epiblast cells in vivo.
Thisi
Whilstin general, in vitro culture of stem cell lines, or their differentiated derivatives, may provide
avreasonable model for studying bottleneck effects, such as those occurring around gastrulation)
when
mtDNA copy number per cell declines to a minimum and replication is initiated (one of theoriginal reasons for the panel suggesting these experiments), they may not offer a fully suitableD
modelof the mtDNA replicative dynamics that obtain during embryonic/fetal development iniutero. In summary, although experiments with ES and other cells cultured in vitro may be af
tractable
way of modelling the behaviour of human mtDNA in post-implantation embryos, thisfdoes
not mean the experiments are reliable (see section 7 for suggested further research).
D(iv) Differences between haplotypes in mtDNA replication: The panel reviewed data
concerning specific combinations of mtDNA haplotypes for evidence that one is preferentially
amplified over the other. Kang et al. (2016), reported that 3 of 18 (17%) ES cell lines derived fromblastocysts produced by MST displayed reversion to the carryover mtDNA haplotype. It seems
unlikely that this is related to the presence of defective mitochondria because it occurred with ES
cell lines derived from two control MST blastocysts as well as for one mutation carrier MST
blastocyst. From the data in their manuscript, it is possible that U5a mtDNA tended to
predominate over H1b and perhaps other haplotypes, but this appears to be independent of thenumber of SNP differences between the two haplotypes.
Yamada et al. (2016) also found that some haplotype combinations were associated with reversal
or instability, although the drift was consistent with some combinations and random with others(see above); however, they were unable to offer a mechanistic explanation for this. The
differences in mtDNA sequence between the various haplotypes were noted, but again, no
obvious explanation was identified.
Similarly, in the one case of reversion seen in an ES cell line by Hyslop et al. (2016) following
PNT, the karyoplast and cytoplast mtDNAs were within the same haplogroup, whereas
combinations of different haplogroups did not show this effect. An additional complication, which
could affect whether genetic drift is seen or not, might involve interactions between specific
mtDNA haplotypes and the maternal and/or paternal nuclear genome. This has been proposed as
the reason underlying the drift seen in experiments where heteroplasmy has been introduced into
mouse and macaque embryos (Johnston et al., 2014; Lee et al., 2012). But the mouse datasuggest that drift is positively correlated with genetic distance between mtDNA haplotypes, so thisis unlikely to explain reversion observed specifically in this human ES cell line. In addition, in the
SCNT experiments of Yamada et al. (2016), where the same nuclear DNA and mtDNA haplotype
was used, the outcome with respect to drift was inconsistent. Moreover, they could detect noeffects on mitochondrial function that correlated with mtDNA haplotype and the nuclear genome.
If it is not the overall differences between mtDNA sequences that are responsible for reversion,another possible cause is variation within the displacement-loop (D-loop) region, in sequencesthat interact with the mtDNA replication machinery. Human mtDNA replication is not fully
understood59,60, but it is known to involve the nuclear-encoded mtDNA polymerase γ (Polγ, the59 Young MJ, Copeland WC. Human mitochondrial DNA replication machinery and disease. Current opinion in genetics &development. 2016 Jun 30;38:52-62.

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POLG product) and several other associated proteins, which interact with specific sequences
within the D-loop together with an RNA primer. In human mitochondria these RNA primers occur
at very low frequency, but evidence suggests that they are transcribed by the nuclear-encoded
mitochondrial RNA polymerase (mtRNAP or POLRMT). This normally directs polycistronictranscription from H-strand and L-strand promoters located in the mtDNA control region, whereas
the primers arise from a switch in its activity that causes RNA transcripts to remain bound to thetemplate as discontinuous but persistent RNA:DNA hybrids, termed ‘R-loops’.
Variation in the sequences to which the replication machinery binds, in the RNA primer
sequences or the transcription initiation sequences to which POLRMT interacts, are all likely toaffect the rate of mtDNA replication (e.g. see: Tan et al., 2016)61. Indeed, mutations within thesesequences are often associated with certain cancers or other pathologies in which mtDNA copy
number is dysregulated62. Epigenetic modifications can also have an effect: demethylation of Dloop sequences has been associated with increased mtDNA copy number in colorectal cancer63,
whereas methylation was linked with reduced copy number in obesity64. However, it is not clear
how these differences in methylation occur, or whether they are a cause or consequence withrespect to altered mtDNA replication.
Whether or not reversion occurs could in theory be associated with allelic variants in the nuclear
genes encoding the factors involved in mtDNA replication, which may affect how their protein
products interact with haplotype-specific D-loop sequences, allowing one to replicate
preferentially over the other. However, Yamada et al. (2016) found no evidence for such mitonuclear interactions.
In order to account for reversion in ES cells in their study, Kang et al (2016) focussed on thehighly polymorphic D-loop region called conserved sequence box II (CSBII). They examined a
CSBII sequence polymorphism (G5AG7) known to be associated with altered efficiency of
mitochondrial transcription termination and replication primer generation. When they analysedmaternal and donor mtDNA in a total of 26 ES cell lines, comprising 18 different haplotype
combinations, they noted that two lines exhibiting reversion (ST-ES7 and ST-ES8) carried donor
mtDNA with a G5AG8 polymorphism and maternal mtDNA of G6AG8. Using in vitro assays they
showed that the G5AG8 polymorphism of donor mtDNA resulted in a 4-fold reduction in
replication primer synthesis. They conclude that the effect of such a D-loop polymorphism(G6AG8), arising from the addition of just a single guanosine residue, may be to confer areplicative advantage to mtDNA harbouring it. Two other ES cell lines showing reversion did notreveal CSBII polymorphisms in donor/maternal mtDNA, but a number of other D-loop
polymorphisms in the core TAS region were detected, and the authors speculate that these may
also contribute to replication bias of a particular mtDNA haplotype over another.
It is worth noting that in cases of heteroplasmy for mutant and normal mtDNA not generated by
MST or PNT, i.e. where there is no heterologous mtDNA nor haplotype mismatch, drift towards
60 Ciesielski GL, Oliveira MT, Kaguni LS. Chapter Eight-Animal Mitochondrial DNA Replication. The Enzymes. 2016 Dec

31;39:255-92.
61 Tan BG, Wellesley FC, Savery NJ, Szczelkun MD. Length heterogeneity at conserved sequence block 2 in humanmitochondrial DNA acts as a rheostat for RNA polymerase POLRMT activity. Nucleic Acids Research. 2016 Sep 19;44(16):781729.
62 Damas J, Samuels DC, Carneiro J, Amorim A, Pereira F. Mitochondrial DNA rearrangements in health and disease—acomprehensive study. Human mutation. 2014 Jan 1;35(1):1-4.
63 Gao J, Wen S, Zhou H, Feng S. De-methylation of displacement loop of mitochondrial DNA is associated with increased
mitochondrial copy number and nicotinamide adenine dinucleotide subunit 2 expression in colorectal cancer. Molecular medicine
reports. 2015 Nov 1;12(5):7033-8.
64 Zheng LD, Linarelli LE, Liu L, Wall SS, Greenawald MH, Seidel RW, Estabrooks PA, Almeida FA, Cheng Z. Insulin resistanceis associated with epigenetic and genetic regulation of mitochondrial DNA in obese humans. Clinical epigenetics. 2015 Jun
10;7(1):1.

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higher levels of one has also been noted to occur during development after PGD. For example, inthe pregnancy reported by Treff et al., (2012), the 12% heteroplasmy level for the m.3243A>G
mutation detected by PGD rose to 52% at 1.5 months after birth46. This suggests that chance
effects resulting in genetic drift can occur. These effects could exacerbate an initial bias caused
by preferential mtDNA replication.
In conclusion, the panel views the stability of the mtDNA profiles found in most of the stem cell
cultures generated from human MST/PNT embryos discussed above as reassuring, but theunusual reversion (upward drift) in the levels of carried-over mtDNA seen in a minority of ES celllines after more or less prolonged culture, is of concern. However, its relevance to what might
happen in human embryos and fetuses in utero following MST/PNT is still unclear. It is notablethat Zhang et al. (2016a) found no evidence of reversion in the two fetuses studied after PNT(which was carried out in an attempt to treat apparent infertility, not to avoid transmission of
mitochondrial disease), nor was there obvious reversion in multiple neonatal tissues following thefirst case of MST reported in recent abstracts10.
From a consideration of all the recent data made available to us concerning reversion of
mtDNA in vitro, and in particular the study of Kang et al (2016), it seems theoreticallypossible that variations in the sequence of the D-loop may confer a replicative advantage
to one haplotype over another in specific combinations. However, further research is
required, particularly because the numbers of MST- and PNT-derived embryos studied so
far remains rather too low to conclusively rule out the other potential mechanisms
discussed above, and, of course because there could be others that have not been
considered here. Finally, the panel emphasises the importance of systematically collecting
data from the long-term follow-up of any pregnancies and subsequent births created by
MST/PNT, in order to establish potential risks to any children born as the result oftreatment.

In 2014, the panel discussed the strategy of transferring only XY (male) embryos following
MST/PNT, to ensure no carryover of maternally inherited mutated DNA to subsequent
generations. In 2014, it did not support such a proposal, even though it would avoid thepossibility of trans-generational inheritance of pathogenic mtDNA, as well as circumvent
objections made by some that the techniques are a form of germ line genetic alteration.
Selecting only XY embryos for transfer would require PGD, an additional step that is likely tocompromise early development of already manipulated embryos; moreover, it would (onaverage) immediately reduce by half the number of embryos available for transfer. This would
decrease the efficiency of the techniques and make it likely that patients would have to undergo
repeated cycles of treatment, which may never be successful. Determining embryo sex after
implantation would require termination of normal XX (female) embryos as discussed as part of
the Nuffield Council on Bioethics ethical review of mitochondrial donation techniques in 201265.

65 Nuffield Council on Bioethics. (2012). Novel techniques for the prevention of mitochondrial DNA disorders: an ethical review.

Retrieved August 22, 2016, from http://nuffieldbioethics.org/project/mitochondrial-dna-disorders.

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There already exists an accepted precedent for assisted reproduction with predictable,deleterious genetic consequences for the next generation: when ICSI is used to overcome maleinfertility in patients with a Y chromosome defect, any son born as a result will carry the same
defect, and ICSI will be required for him to have a child. This will be true for all subsequent
generations of males of this lineage. In contrast, for MST and PNT, the risks of mitochondrial
disease in the next generation are likely to be low. The subsequent use of PGD in this next
generation, to select embryos with very low or no levels of abnormal mtDNA, may rid all
subsequent generations of the need to have interventions to avoid mitochondrial disease.
During this current review in 2016, the panel noted the recent report on mitochondrial
replacement techniques (MRT) from the National Academies of Sciences, Engineering andMedicine19, which had been commissioned by the US Food and Drug Administration (FDA). A
committee was convened to examine and analyse issues relating to techniques for avoiding some
types of inherited mitochondrial DNA (mtDNA) diseases and make recommendations for further
clinical investigations.
This report recommended limiting clinical research to the transfer of MRT XY (male) embryos, so
as to avoid heritable genetic modification, with no transfer of female embryos until there “is clear
evidence of safety and efficacy from male cohorts, using identical MRT procedures, regardless of
how long it took to collect this evidence”. The panel had difficulty in supporting this view due to therequirement for additional manipulation of the embryo, the reduction in embryos available fortransfer, the deliberate exclusion of normal XX (female) embryos and the impracticalities ofattempting to deliver absolute safety. Importantly, it should be noted that under the HFE Act, sex
selection is only permitted for sex-linked diseases, which do not include diseases due tomutations in mtDNA.
In 2016, the panel agreed that their view on this matter has not changed and therefore they
do not support any proposal to select XY (male) embryos only.

In 2014 the panel received little new evidence in relation to this area. However, alternative
methods to prevent abnormal activation were described in a series of recent papers reporting
SCNT with human oocytes. These made use of low levels of caffeine, putatively as a protein
phosphatase inhibitor, in the medium during spindle removal and fusion of the somatic cell. In
addition, it was also clear that care needs be taken to prevent fusion of polar bodies back into
the oocyte or zygote, as this could produce aneuploid embryos66,67,68

66 Yamada M, Johannesson B, Sagi I, Burnett LC, Kort DH, Prosser RW, Paull D, Nestor MW, Freeby M, Greenberg E, Goland

RS. Human oocytes reprogram adult somatic nuclei of a type 1 diabetic to diploid pluripotent stem cells. Nature. 2014 Jun
26;510(7506):533-6.
67 Chung YG, Eum JH, Lee JE, Shim SH, Sepilian V, Hong SW, Lee Y, Treff NR, Choi YH, Kimbrel EA, Dittman RE. Humansomatic cell nuclear transfer using adult cells. Cell Stem Cell. 2014 Jun 5;14(6):777-80.
68 Tachibana M, Amato P, Sparman M, Woodward J, Sanchis DM, Ma H, Gutierrez NM, Tippner-Hedges R, Kang E, Lee HS,
Ramsey C. Towards germline gene therapy of inherited mitochondrial diseases. Nature. 2013 Jan 31;493(7434):627-31.

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Human Fertilisation and Embryology Authority

In 2016 the panel received no new evidence specifically related to this topic. However, thesuccess rates described in Kang et al. (2016) for blastocyst generation and levels of
aneuploidy suggest distinct improvements have been made in this area.

During the 2014 considerations an oral presentation was given to the panel by Dr Iain
Johnston,of Imperial College London and subsequently a manuscript was provided (nowpublished as Burgstaller et al., 2014). The group explored data that investigated mtDNA
segregation and its possible dependence on genetic distance between distinct haplogroups,
where mito-nuclear interactions may, theoretically, play an important role in the mechanism.
These experiments were designed to explore, in an artificially induced heteroplasmic situation in
mice, whether some mtDNA haplotypes experience a selective advantage over others in certain
tissues and over time. The notion that this might happen was first highlighted by Jenuth et al.,(1996) who investigated mice carrying mtDNA from both NZB and BALB/c strains that had been
derived from founders made by ooplasm transfer. The panel also considered the separate issueof whether it is possible that an incompatibility could arise between nuclear and mitochondrial
genomes in an embryo generated by MST or PNT.
In a submission and presentation from Dr Edward Morrow, University of Sussex, some of theirgroup’s concerns relating to the safety of mitochondrial replacement techniques, expressed in
the recent article by Reinhardt et al., (2013) and in a follow-up manuscript submitted as
evidence32, were revisited. The presentation highlighted their views on nuclear-mtDNA
interactions and the anterograde and retrograde signalling between these DNA genomes,
emphasising this signalling cross-talk and the possible effects on oxidative phosphorylation,
metabolic pathways and other processes that they propose might result from disrupting theseinteractions. Discussions concerning these two separate issues relating to mito-nuclear
interactions are detailed in the 2014 report (page 30-31).
In summary, the panel reviewed two separate arguments for considering haplotype/haplogroupmatching. The concern of Morrow and co-workers is based on evolutionary arguments thatsome nuclear-haplotype combinations can lead to a failure of co-evolved mito-nuclear
interactions with adverse consequences. They propose haplotype, or at least haplogroup,
matching to reduce the risk of any such effect occurring. Their arguments did not address
pathogenic (disease pre-disposing) mutant mtDNA per se. Moreover, Morrow and colleagues
did not address the possibility that an exchange to a different haplotype/haplogroup might, insome cases, confer advantages69. The panel considered that the likelihood of such hypotheticalproblems occurring would be low. Given the high degree of variation in the nuclear genome that
can have an impact on embryo fitness, whether this is due to effects on mitochondrial function ornot, the consequences of pursuing any strategy involving matching are likely to be minimal. The
Drosophila research cited in the 2014 report70 used inbred strains, which makes it more likelythat any effects of a mismatch will be detected, whereas humans are much more outbred so that69 Latorre-Pellicer A, Moreno-Loshuertos R, Lechuga-Vieco AV, Sánchez-Cabo F, Torroja C, Acín-Pérez R, Calvo E, Aix E,

González-Guerra A, Logan A, Bernad-Miana ML. Mitochondrial and nuclear DNA matching shapes metabolism and healthy
ageing. Nature. 2016 Jul 28;535(7613):561-5.
70 Innocenti P, Morrow EH, Dowling DK. Experimental evidence supports a sex-specific selective sieve in mitochondrial genome
evolution. Science. 2011 May 13;332(6031):845-8.

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such effects are unlikely to be detectable.
Data from Johnston et al.71,72 raised the concern that, after MST or PNT, the now rare mtDNA
variant carrying the mutation would preferentially expand (due to some unknown mechanism) insome tissues. This could occur if that variant possesses some "selectable" advantage in thosetissues in comparison to the mtDNA variant provided by the donor oocyte. If a selective
mechanism were operating, it could do so within (mtDNA competition) or between cells (cellcompetition) in the developing embryo or newborn. Any post-natal manifestation following MST
or PNT might be deleterious for the child and/or subsequent generations if the child were a girland the selection process operated in the germ line.
This may be different from the considerations of Morrow et al., which concern failure of mitonuclear interaction; but any selective advantage conferred by a mitochondrial variant could
depend on mito-nuclear interactions. Haplotype/haplogroup matching was proposed to remove
any putative selective advantage. In addition, it might one day be possible to choose a donor
carrying a haplogroup that has a selectable advantage (considered in the context of the child'snuclear DNA), which would beneficially out-compete the variant carrying the mutation. However,as with Morrow et al., the arguments of Johnston et al. are based on an inbred animal model.
The outbred nature of humans makes it much less likely that any effect on mtDNA segregation
would occur73 or, if it did, that any reliable correlation could be made.
In conclusion, the panel considered evidence presented concerning the two issues relating to
mtDNA haplotypes: (i) that mito-nuclear mismatch as a result of MST or PNT might lead to
unexpected adverse effects on the offspring - albeit that this does not seem to occur naturally,and (ii) possible segregation in favour of any carried-over mutant mtDNA, possibly exacerbatedby unusual mito-nuclear interactions. Whilst the panel acknowledges that haplogroup matchingof donors could be a possible precautionary step that might ameliorate such effects should they
operate in humans, they appreciate that it may not always be possible, especially when close
relatives might also be at risk of transmitting the mtDNA mutations, or when the patient has an
unusual haplotype compared with the possible donor population. Further evidence would beneeded to understand whether and how the proposed effects might be operative.
In 2014 the panel recommended that consideration should be given to mtDNA haplogroupmatching when selecting donors, although the panel considered that the risks of not doing sowould be very low, and acknowledged that there may be practical factors preventing it. In
addition, the panel recommended that the follow-up of children born as a result of these new
methods should include assessment of whether any preferential expansion of the mitochondria
carrying the mutations occurs over time, and comparison between the general state of health ofthe child and the degree of haplotype mismatch. This follow-up could help to inform future
decisions about matching.

During this 2016 review, the panel discussed issues arising from possible haplotype mismatchingand noted several contributions to the topic of how conserved mito-nuclear compatibility may bein the human population:
71 Burgstaller JP, Johnston IG, Jones NS, Albrechtova J, Kolbe T, Vogl C, Futschik A, Mayrhofer C, Klein D, Sabitzer S, Blattner

M. MtDNA segregation in heteroplasmic tissues is common in vivo and modulated by haplotype differences and developmental
stage. Cell reports. 2014 Jun 26;7(6):2031-41.
72 Burgstaller JP, Johnston IG, Poulton J. Mitochondrial DNA disease and developmental implications for reproductive strategies.
Molecular human reproduction. 2015 Jan 1;21(1):11-22.
73 James JE, Piganeau G, Eyre‐Walker A. The rate of adaptive evolution in animal mitochondria. Molecular ecology. 2016 Jan
1;25(1):67-78.

Scientific review: 2016 update

Human Fertilisation and Embryology Authorityi) Ma et al. (2015) used somatic cell nuclear transfer from patient fibroblasts, harbouringmitochondria associated with Leigh syndrome and MELAS, to donor oocytes (with normal
mitochondria) and derived pluripotent stem cell lines (PSC) from these. Patient mtDNA
haplogroups and donor haplogroups varied. They were able to derive PSCs containing noresidual patient-derived mtDNA and showed that transcriptional and epigenetic profiles of thesewere similar to conventional ES cells. Despite haplogroup differences between patient and donor,
at 47 nucleotide positions of the mtDNA, normal metabolic function in PSCs was observed. They
conclude that normal nuclear-to-mitochondrial interactions are highly conserved within species.
ii) In their study of MST followed by parthenogenetic activation of reconstructed oocytes, Yamada
et al. (2016) examined stem cells that had identical nuclear genomes, but were homoplasmic foreither the L0 or K1 haplotypes. No significant differences in mitochondrial function were observed.
Yamada et al. (2016) conclude that the maternal nuclear genome is fundamentally compatible
with different mtDNA haplotypes. They also note that this is not unexpected since maternal
nuclear alleles may re-segregate with a new mtDNA haplotype after transmission through a son.
iii) In their examination of the transcriptomes of cells isolated from ePNT and control blastocysts,
Hyslop et al. (2016) examined expression of mtDNA-encoded oxidative phosphorylation(OXPHOS) genes. They note that, although there was wide variation in mtDNA OXPHOS gene
expression within and between ePNT and control blastocysts, samples from both groups
clustered together after unsupervised hierarchical clustering, irrespective of whether the
karyoplast and cytoplast contained the same or different mtDNA haplotypes. They conclude thatswitching nuclear genomes does not result in a detectable effect on mitochondrial gene
expression.
iv) In their study of MST followed by normal fertilisation of reconstructed human oocytes, Kang etal. (2016) analysed development of MST embryos with differing degrees of divergence between
the karyoplast and cytoplast mtDNA haplotypes, ranging from closely related (six single
nucleotide polymorphisms (SNPs)), to medium (33 SNPs) to distant (57 SNPs). Fertilisation andblastulation rates were similar among all three groups. They conclude that MST does notadversely affect mtDNA-nuclear interactions during human embryonic development in vitro.
The above reports, all investigating the possibility of mito-nuclear incompatibility in humanembryo-derived cell lines, embryo-derived cells or whole embryos following mitochondrial
donation techniques conclude that such a possibility remains theoretical. All focus on in vitro
studies, and thus their relevance to the whole adult organism is unclear. But they are notable instudying this possibility in the context of human outbred genomes and conclude that suchgenomes can robustly interact with a variety of common, but genotypically distinct, mtDNA
haplotypes that presumably evolved to be so compatible. The panel also continued to monitor theuse of model organisms to address possible mito-nuclear incompatibility.
Latorre-Pellicer et al. (2016) studied mice in which the C57BL/6 (B6) nuclear genome is combinedwith either the B6 mitochondrial genome as usual (B6-C57), or the mitochondrial genome fromthe NZB/OlaHsd strain of mouse, to yield the conplastic strain, B6-NZB. (N.B. this was carried out
by repeated backcrosses, not by MST or PNT.) The level of divergence between the B6 and NZB
mtDNA genotypes is reported to be comparable to that between human Eurasian and African
mtDNAs. They characterised both strains throughout their lifespan using a range of phenotypic
parameters. Unexpectedly, the conplastic B6-NZB strain exhibited an extended median lifespanof 16%. Two-year old B6-C57 mice manifested more signs of ageing than conplastic counterparts.
Respiration in B6-C57 mice also declined steadily with age, whilst B6-NZB remained constant
between days 20 and 300. The authors conclude that NZB mtDNA promotes healthier ageing in
the B6 nuclear background. They also report that mtDNA haplotype influences mitochondrial
proteostasis and reactive oxygen species generation, insulin signalling and obesity. On the basis

Scientific review: 2016 update

Human Fertilisation and Embryology Authorityof these observations, the authors suggest that consideration should be given to matching the
mtDNA of patient and mitochondria donor if performing mitochondrial donation.
While these data are interesting, the relevance to humans, who are outbred and undergo regularexchange of mtDNA haplotype and nuclear genomes, is unclear; these points were made in the2014 report. But in addition, the finding that there is a significant advantage to B6 mice inexchanging their mtDNA to that of NZB adds an additional complication. This could be seen not
as an argument for matching haplotypes, but for deliberately searching for one that is
advantageous. Moreover, because the reciprocal conplastic strain (NZB-C57) was not generated,
it is not known if these would have fared less well or have had the same or other advantages.
Ma et al. (2016) performed reciprocal mitochondrial donation using PNT between two subspecies
of mouse that have diverged from a common ancestor for over 500,000 years, B6 and PWD. As
expected, these strains are highly divergent in their mtDNA genotypes, differing by 391 SNPs
(2.4% of the mtDNA genome). Remarkably, post-implantation development of embryos with a B6
nuclear genome and PWD mtDNA (B6-PWD) was not significantly different to controls. Malefertility in adults was reduced, but not in females. In contrast, embryos with PWD nuclear DNA
and B6 mtDNA (PWD-B6) were much less viable: only one live pup out of 359 transferred
blastocysts was obtained; the rest were stillborn. This lethality likely reflects incompatibility
between PWD nuclear DNA and B6 mtDNA. The authors propose that these data constitute direct
evidence that mtDNA genotype divergence plays a major role in reproductive isolation.
The significance for humans of the mtDNA-nuclear DNA incompatibility revealed by this study isunclear. Average mtDNA haplotype divergence in humans is bimodal, with a main peak at 45
SNPs and a secondary peak at 100 SNPs74. The PWD-B6 mtDNA divergence (391 SNPs) is far
greater - and it is striking that B6-PWD conplastic mice are still viable despite this. Nevertheless,the authors suggest that matching haplotypes of the patient and mitochondrial donor in clinicalapplication of mitochondrial donation would be judicious. It is also worth noting that inbred mousestrains are commonly used because they allow the consequences of particular mutations to bestudied without additional confounding genetic variation, and because phenotypes in general aremuch more highly penetrant on such backgrounds. This is probably due to the fact that the almost
all loci in such mice are homozygous at all nucleotide positions, apart from those affected by denovo mutation. Outbred genomes, such as that of humans, provide a greater degree of functional'buffering' due to common heterozygosity.
The panel’s view on mito-nuclear interactions has not been altered by recent data. An
argument can be made for deliberately choosing a donor haplotype (or at least D-loop
sequence) based on its ability to out-compete that of the patient to avoid any possibility ofreversion. However, this would require further research and an understanding of theoutcome of clinical applications of the methods. The panel maintains its earlier conclusionthat consideration should be given to haplotype/haplogroup matching based on theoutcome of future research and clinical experience.

74 Blanco R, Mayordomo E, Montoya J, Ruiz-Pesini E. Rebooting the human mitochondrial phylogeny: an automated andscalable methodology with expert knowledge. BMC bioinformatics. 2011 May 19;12(1):1.

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In 2014, the panel received no new evidence on epigenetic effects to alter their conclusions
reached in 2013. One of the experts the panel consulted during the 2014 review highlighted thatthe nuclear DNA of mothers carrying a proportion of mutant mtDNA could undergo some
remodelling of genes (e.g. epigenetic modification), notably those involved in mitochondrial
function, with the result that these might then compensate for the deficiency in the mutated geneon the mtDNA and thereby reduce the phenotypic consequences of the mutant mtDNA. Suchepigenetic effects might also affect subsequent mito-nuclear communications in a novel way;
however, to be relevant, this would have to occur in the germline and particularly in oocytes,
which is unlikely given the rather inactive state of mitochondria in these cells. The experts
considered this to be an unlikely eventuality, but of low risk if it did occur.
The panel also considered the possibility of epigenetic or other deleterious effects on embryos
as a result of oxidative stress during the mitochondria replacement procedures. They concluded
that there is substantial evidence that mitochondria with different haplotypes vary in theirtolerance to oxidative stress; however, this is likely to be more relevant in mature tissues, where
mitochondrial function is critical, and less of a concern to early embryos in culture.
The panel’s view on potential epigenetic effects remained unchanged from thoseexpressed in previous reports. Any risks with respect to MST and PNT would appear to below. However, the panel recognised the need to monitor research in this area.

Scientific review: 2016 update

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It seems clear from the experiments of Hyslop et al. (2016), Yamada et al. (2016) and Kang et al.
(2016) that some clones of stem cells derived from human embryos constructed by MST or PNT
can accumulate significantly elevated levels of karyoplast-associated mtDNA with increased
passaging. Should this phenomenon be replicated in utero following clinical application of
mitochondrial donation, it could lead to the birth of an affected child. Without a definitive
mechanism to account for this reversion, the panel considered it difficult to determine the risk ofoccurrence in vivo. It may be that these effects were peculiar and specific to embryonic stem cell
populations and their differentiated derivatives that were being used in vitro as a model of thedeveloping embryo. Or, it may be that they were effects that might occur in embryos developing inutero in their usual three dimensional morphology and particular niche, as a result of carryover ofkaryoplast-associated mitochondria that preferentially replicate and cause unexpected
symptomatic heteroplasmy. This theoretical possibility was raised in section 3 of the 2014 report.

In considering the potential for adverse effects of pathogenic mtDNA carryover following MST or
PNT, the panel made comparisons with mutation levels (heteroplasmy) in the embryo when PGD
is undertaken for mitochondrial disease (see Table 1). It is apparent from the published literature
discussed below, and from interviews conducted by the panel, that decisions about safe levels ofheteroplasmy vary widely between clinics practicing PGD for mitochondrial disease. Dependingon the specific mutation, it is not uncommon for there to be few, if any, tested embryos with
undetectable levels of heteroplasmy; most embryos contain ‘low’ levels of pathogenic mtDNA
within a range purported to have a low or reduced likelihood of manifesting symptoms in the
future child75, but these levels are usually higher than the levels of carryover achieved (or limits
proposed) following MST or PNT. Moreover, there are cases with homoplasmy or very high levelsof heteroplasmy, where PGD can never give an embryo with acceptable levels of pathogenic
mtDNA.
Whereas some clinics use a theoretical consideration of a general level of mutant heteroplasmy
below which symptoms are unlikely in order to set a maximum for transfer, it is clear that the ‘safe’level will vary with the nature of the mutation and the particular clinical features it gives, and thelikelihood that the symptoms will be serious46. Estimates of these ‘safe levels’ may well be
improved as more information becomes available with use of PGD for mitochondrial disorders,
although this will take time to accumulate. In addition, some centres prefer to take into accountthe patients’ wishes, especially when there are no ‘safe level’ embryos to replace. One view isthat it might even be acceptable to replace an embryo with a higher-than-threshold level of mutant
heteroplasmy, recognising that, even at such levels, this would nevertheless represent a risk
reduction from the high levels that would probably be present should the couple have decided toreproduce naturally12.
75 Hellebrekers DM, Wolfe R, Hendrickx AT, de Coo IF, de Die CE, Geraedts JP, Chinnery PF, Smeets HJ. PGD andheteroplasmic mitochondrial DNA point mutations: a systematic review estimating the chance of healthy offspring. Human
reproduction update. 2012 Jul 1;18(4):341-9.

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Table 1: Mutation levels in live births following PGD and MST for mitochondrial disease76, 77
PGD data (2005-2016)
Mutation

At biopsy

At birthComments

Reference

1

m.8993T>G

0% & 0%0%First report. Two embryos
transferred

Steffann et al., 2006

2

m.8993T>G

2.5%

4%

3-5% cord blood &placenta; buccals 5% at
age 4½y

Thorburn et al 2010*

3

m.3243A>G

5% & 13%

5%

Two embryos transferred;
15±5% placenta, 5±1%
cord blood

Monnot et al., 2011

4

m.3243A>G

12%

15%

47% blood, 52% urine @1½m; 46/42%@18m

Treff et al., 2012/
Mitalipov et al., 2014

5

m.8993T>G

0%

0%'Healthy son', no further
details

Sallevelt et al., 20136

m.8344A>G

53% & 59%

63%

Two embryos transferred;
no further details

Steffann et al., 2014

7

m.3243A>G

0%0%Male; measured in cord
blood, urine, saliva

Heindryckx et al.,20148

m.36**G>A

2%

7%

Female, measured buccal
and urine cells

Newcastle group
2016*

9

m.83**A>G48%

Not
Male; <60% generally
available asymptomatic

Newcastle group
2016*

10

m.130**T>C1%0%

Male; undetectable in cordand peripheral blood

Newcastle group
2016*

11

m.101**T>C

1%

1-2%

Male; cord blood

Newcastle group2016*

Reference

MST data (2005-2016)

1

Mutation

At biopsy

At birthComments

8993T>G

5.7%

1.6%

‘Healthy’ male; 1.60 ±Zhang et al., 2016b,
0.92% measured in buccal Liu et al., 2016***
cells, hair follicles, foreskin,urine, placenta, amnion,
cord blood, and cord tissue.

76 Each row corresponds to one live birth.
77 * personal communication** characters hidden to respect confidentiality*** abstract.

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The particular mutation has a bearing on what is considered a ‘safe’ level. Whilst embryos from
3243A>G carriers manifest a full range of mutation loads and thus are associated with more in theintermediate, and potentially symptomatic range, and have fewer embryos with low or absent
levels, other mutations (e.g. 8993T>G) show skewed distributions with embryos tending to fall ineither the very high or the very low or absent range, allowing a greater margin of safety in the low
range78,79.
However, there is still some uncertainty when deciding on ‘safe’ levels of mutant mtDNA forembryo transfer, since the actual proportion of pathogenic mtDNA present in the child once born
could be somewhat higher or lower than that predicted from the embryo biopsy. This might be due
to effects of the mitochondrial bottleneck in early development, and the possibility of non-random
genetic drift or preferential amplification of abnormal mtDNA11. The panel is aware of the PGD
case report (Treff et al. and 15; see Table 1, case 4) where levels rose from 12% at blastocyst
biopsy, 15% in buccal cells at birth, to 52% in urine cells at 1½ months of age48. Therefore, in
addition to reviewing published cases, the panel made contact with centres internationally thatpractise PGD for mitochondrial disease in order to ascertain how frequently this happens. The
panel noted that although there seem to have been very few babies born as a result of PGD for
mtDNA mutations (see table 1), in general, heteroplasmy at birth is not very different from that atembryo biopsy, even when levels of mutation load are much greater than the <5% recommended
for PNT and MST. However, it clearly is possible that significant drift can occur after birth, even insituations where the mutation resides in an otherwise genetically homogeneous population of
mtDNA, as illustrated by the case described above.
The possibility that drift similar to that seen in PGD could arise after MST or PNT cannot be
excluded. In addition, as discussed at length above (notably in section 4), there may be othereffects specific to MST and PNT due to the transfer process itself, which may occasionally'activate' the mtDNA that was associated with the karyoplast, or as a result of the highly
asymmetric distribution of karyoplast-associated mtDNA that is generated in the reconstructedembryo, or because of the presence of two different haplogroups/haplotypes, where one has areplicative advantage over the other. In this regard, the panel was mindful of the concerns
expressed in submissions, and implicit in the recent publications, about reversion towards or tothe mtDNA haplotype associated with the karyoplast in a small percentage of stem cell lines.
However, it is noteworthy that in the report of Zhang et al., (2016a), where PNT was undertaken
for treatment of infertility rather than avoidance of genetic disease, examination of the two midtrimester fetuses showed no presence of the maternal mtDNA mutation. Moreover, in the recent
abstracts describing the use of MST for the avoidance of transmission of m.8992T>G10, the reportof low heteroplasmy levels in the baby suggests that substantial reduction of mutant mtDNA by
PNT and MST is possible.
Our understanding of mitochondrial donation techniques will doubtless improve as more clinically
relevant models are developed and further data are obtained from human embryos. It is to be
hoped that this will lead to a mechanistic understanding of likely behaviours of mitochondria and
mtDNA in vivo, and therefore strategies to avoid adverse effects, if necessary. For these reasons,the panel recommends that it would be useful to examine the behaviour of mtDNA in stem cells
derived from PGD embryos (unsuitable for transfer) with low levels of heteroplasmy.

78 Steffann J, Monnot S, Bonnefont JP. mtDNA mutations variously impact mtDNA maintenance throughout the humanembryofetal development. Clinical genetics. 2015 Nov 1;88(5):416-24.
79 Blok RB, Gook DA, Thorburn DR, Dahl HH. Skewed segregation of the mtDNA nt 8993 (TRG) mutation in human oocytes. The
American Journal of Human Genetics. 1997 Jun 30;60(6):1495-501.

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In making a decision about the efficacy and clinical safety of mitochondrial transfer techniques,
the panel noted:(i) PNT methods can now efficiently produce good quality blastocysts based on a number ofcriteria(ii) much higher levels of mutation heteroplasmy are accepted for PGD than are recommended asa target for PNT and MST (<5%)(iii) reversion to the karyoplast-associated mtDNA haplogroup occurred in only a proportion(around 10-25%) of stem cell lines cultured from MST embryos in vitro(iv) even use of PGD may not eliminate transmission of mtDNA mutations, but it is still useful forrisk reduction in the offspring. In addition, the panel considered the recent report of a three-monthold, apparently healthy baby born after MST, with low levels of pathogenic mtDNA in several
tissues, to be reassuring. The panel thus felt that MST and PNT are now at an acceptable stage
for cautious clinical use as a risk reduction strategy in carefully selected cases49: those where
PGD is unlikely to be successful or impossible (e.g. due to high levels of maternal pathogenic
mtDNA), or where PGD has been unsuccessful. In these circumstances the patients would haveto be counselled as to the risks, and offered prenatal testing should they become pregnant.
The panel discussed ways of predicting the phenotype prenatally29,31. Prenatal testing by CVS,
amniocentesis or fetal blood sampling was considered. Amniocentesis may be scientifically
preferable, as the cell types present in amniotic fluid come from all three embryonic lineages,
whereas fetal blood cells are entirely mesodermal in origin, and the risk of miscarriage following
amniocentesis is extremely low. However, the greater yield of cells from CVS compared with thatfrom amniocentesis means that CVS is the preferred option in most diagnostic laboratories. Noneof these sample types for prenatal testing would necessarily be representative of outcome, so it
would be important to counsel patients about the pros, cons and limitations of prenatal testing
before any decisions are made.
Due to the difficulties in predicting whether an embryo will undergo reversion to the mutant
mtDNA haplotype carried over from eggs or zygotes from patients, and the possibility of otherunpredictable outcomes of the treatment, the panel concludes that it would be prudent to restrict
mitochondrial donation treatment to patients whose clinicians believe that PGD is not a feasible
option because of predicted homoplasmy or high levels of heteroplasmy in their embryos. This
restriction may be revised following analysis of data from treatment cycles. Indeed, as the panel
has suggested previously, if MST and/or PNT prove safe and reliable, then they could beconsidered preferable to PGD as a risk reduction strategy for mitochondrial disease, not just in
the children born, but for subsequent generations. The panel also advises that prenatal testingshould be offered to all women undergoing treatment, but recognises that it is unlikely that all
women will accept this offer. Long-term follow-up of the children is strongly recommended.

Scientific review: 2016 update

Human Fertilisation and Embryology Authority

The panel’s view is that the methods of PNT and MST are sufficiently safe to proceed cautiously
and in restricted circumstances. The science in this area has not stood still. Much progress has
been made since the last review in 2014 when the panel offered the view that these methods
were not unsafe but that further research was still needed. Since that time, significant work has
been carried out, both in the basic research field to improve understanding of the biology ofhuman mitochondria, especially in development, and on translational research aimed specificallyat providing further safety information on MST and PNT. The panel’s previous requests for
additional experiments have largely been met and many of the concerns that had been raised are,in its view, even less likely to be a problem.
One new issue that has arisen in the recent work from three groups is reversion towards the
karyoplast-derived mtDNA haplotype after prolonged culture of ES cell lines derived from aminority of embryos made using MST or PNT. It remains unclear whether reversion will occur invivo and be a problem if the techniques are applied clinically. However, this is a potential risk thatthe panel takes seriously and hence it recommends restricted application in the first instance. An
alternative for the panel was to recommend that clinical application should not proceed until there
was better understanding of the causes of the reversion and approaches designed to reduce or
eliminate it. However, the panel felt that this approach was too cautious, especially when the needis real, and the risks may be small and manageable to some extent via prenatal testing, an optionthat may be acceptable to some patients.
As stated in the previous reports and above, complete reassurance will never come fromexperiments conducted in animal models and with human material in vitro. Therefore, it should be
accepted that there will always be some risk and uncertainties associated with the use of MST or
PNT in humans until it is tried in practice80. It is with due consideration of this point that the panel
recommends that techniques to avoid mitochondrial disease (MST and PNT) could beinitially implemented as a risk reduction treatment for carefully selected patients.

For the time being, the panel suggests limiting the scope of MST/PNT treatment to patientsfor whom PGD may be inappropriate and probably unsuccessful, such as women whose
germ line is likely to be homoplasmic or have high levels of heteroplasmy; such treatment will
provide these patients with the choice of an alternative intervention for risk reduction. MST/PNT iscurrently the only option for producing a genetically related child for these patients. The panel
does not recommend that the clinical use of MST or PNT should be extended to otherwise healthy
individuals with fertility problems, because a strong causal link between infertility and impaired
mitochondrial function has not been made; in addition, such clinical use is not legal in the UK.
Pre-treatment assessment would need to take into account the particular mutation involved, theinheritance pattern in the family, the likely clinical manifestations of disease, patient age and theefficacy of any previous treatments such as PGD, and the patient's understanding of the risks andlimitations of what is being offered. If the techniques prove to be safe when used in these80 Bredenoord AL, Braude P. Ethics of mitochondrial gene replacement: from bench to bedside. Bmj. 2010 Nov 8;341:c6021.

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patients, including the absence of any significant reversion to the carried-over mtDNA haplotype,their application could be extended to other patients.

The panel recommends that if mitochondrial donation treatments are implemented, patientsshould be offered prenatal testing following the transfer of any embryo that has beencreated as a result of these techniques; however, it recognises the likelihood that not all
women will accept this offer.
The panel also strongly recommends rigorous long-term follow-up of children born as aresult of these techniques in order to gather further information about safety and efficacy.

As highlighted in the 2014 report, the panel drew attention to the parallels with PGD for mtDNA
mutations in terms of acceptable levels of heteroplasmy. Although the intention of such therapy is
to select embryos for transfer with as low a level of mutant mtDNA as possible to avoid the birth ofa child who would manifest the disease in their lifetime, concerns related to variable segregationof mutant mitochondria in their tissues, and especially their gametes, also apply here.
Hence, the panel recommends that for PGD of mtDNA mutations, clear rules are developedthat establish acceptable levels of mtDNA heteroplasmy, indicating when transfer (or not)of an embryo is advised for each disease and each mutation, managed by the specialist
clinical team in conjunction with their patients.
As with MST and PNT, follow-up of children and their offspring is strongly recommended
for individuals arising from PGD for mtDNA mutations.

The panel continues to recommend that consideration is given to mtDNA haplogroup13
matching as a precautionary step in the process of selecting donors. Although the panel
believes that any risks associated with a mtDNA-nuclear DNA mismatch remain theoretical, it
recommends that when these techniques are used clinically, the latest evidence regarding how
mtDNA haplotypes affect mitochondrial-nuclear (mito-nuclear) interactions, including replicative
behaviour of mtDNA, should be considered in order to inform the donor selection process. Suchevidence might even indicate the selection of a specific, unmatched donor in any given case.
Whatever decision is made, the panel recommends that haplotype information on therecipient and the donor is recorded.

Basic research is still needed to explore how the mitochondrial bottleneck functions, addressing
the critical parameters involved in the segregation of normal and any specific abnormal
mitochondria amongst cell types in humans, because this is generally not well understood. For
example, in the long term it may be possible to influence or control replication of abnormal mtDNA

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in the early embryo to affect its segregation or inheritance in subsequent development81. This isdiscussed in more detail in section 7.
Our understanding of mitochondrial donation techniques will doubtless improve as more clinically
relevant models are developed and further data are obtained from human embryos. It is to be
hoped that this will lead to a mechanistic understanding of likely behaviours of mitochondria and
mtDNA in vivo, and therefore strategies to avoid adverse effects, if necessary. For these reasons,the panel recommends that it would be useful to examine the behaviour of mtDNA in stem cells
derived from PGD embryos (unsuitable for transfer) with low levels of heteroplasmy.

81 Gammage PA, Rorbach J, Vincent AI, Rebar EJ, Minczuk M. Mitochondrially targeted ZFNs for selective degradation ofpathogenic mitochondrial genomes bearing large‐scale deletions or point mutations. EMBO molecular medicine. 2014 Apr
1;6(4):458-66.

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In reaching a view that MST and PNT are sufficiently safe that they could now be offered tocertain patients, the panel also recognises that further research is still desirable. Together withsafety and efficacy data from first attempts, the data generated by such future research may
result in more patients benefitting from these techniques. It is also important to obtain more basic
knowledge about the biology of mitochondria in humans and model systems. This may shed lighton the safety and efficacy of mitochondrial donation, lead to novel ways to prevent mitochondrial
diseases in offspring, and/or enable treatment for individuals already affected by them. The
suggestions below are not intended to be exhaustive.

Although the panel continues to consider these unlikely based on the evidence it has seen, any
deleterious or beneficial effects of altering mito-nuclear interactions should be explored, where
possible, in scenarios as relevant as possible to human physiology. The panel recommend thatgenome-wide association studies (GWAS) and other large-scale human genome sequencing
projects that are linked to health, which may have been initiated for other reasons, should include
an analysis of mtDNA sequences and how these are inherited. Detailed knowledge of thestructure of mitochondrial complexes involved in OXPHOS could also be used to help understandand perhaps even predict how nuclear and mtDNA-encoded components interact and how
variations in the gene sequences might have a bearing on their function82

Existing whole genome sequencing projects linked to health and lifestyle records for broad humanpopulations, such as the 100,000 genomes project83 provide useful information relevant to clinicalmanifestation of mitochondrial disease and options for treatment or its avoidance. In addition,
focussed studies involving whole genome sequencing of patients with mitochondrial disease andunaffected family members (as well as comparisons with the general population) might reveal
whether there are any predisposing nuclear genomic alleles or specific combinations of nuclear
and mtDNA variants that are more likely to be associated with disease (with the caveat that
numbers may be too low to give robust answers). A recent review31 by Sallevelt et al (2016)
reported that in 24.6% of patients with (likely) pathogenic mtDNA point mutations, the mutation
had arisen de novo. This group presents an opportunity to assess whether one or more nuclear
genome sequences, perhaps in combination with specific mtDNA variants, predispose to mtDNA
mutations. Genes involved in mtDNA replication or which lead to high levels of ROS are likely
candidates – indeed they are already known to be involved: one of the six de novo cases
discussed by Sallevelt et al. (2016) carried a POLG mutation.

82 Fiedorczuk K, Letts JA, Degliesposti G, Kaszuba K, Skehel M, Sazanov LA. Atomic structure of the entire mammalian

mitochondrial complex I. Nature. 2016 Sep 5.
83 The 100,000 Genomes Project. Accessed at: https://www.genomicsengland.co.uk/the-100000-genomes-project.

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It is not yet clear whether the reversion phenomenon is specific to ES cells in culture and/or
whether it would be seen when heteroplasmy involves the same haplotype (i.e. after mtDNA
mutation) or if it only occurs when heteroplasmy involves a mix of two (or more) mtDNA
haplotypes (e.g. after MST, PNT or SCNT). Even if it turns out to be mostly specific to ES cells, it
is still worth investigating if it reveals information about mtDNA bottlenecks, upward drift in the
proportion of mutant mtDNA often seen with ageing in specific cell types, and the effects ofspecific variants on mtDNA dynamics (such as replication) in combination with specific nuclear
alleles. Some studies have been performed on iPS cell lines derived from patients withheteroplasmy23, but it would also be worth studying ES cell lines derived from embryos known tocarry different levels of heteroplasmy after PGD. It would also be possible to create large panels of
ES or iPS cell lines with different combinations of mtDNA haplotype by making cybrids (fusing
enucleated cells of one haplotype with ES/iPS cells of another).

The recent demonstration that it is possible to derive functional mouse oocytes entirely in vitro
from ES cells and iPS cells suggests several future prospects that might help avoid mitochondrial
disease84. The panel acknowledges that the generation of human oocytes in vitro will pose many
problems, not least how to deal with issues of timing, where development of the first oocytes to a
fully grown stage in humans usually takes over a decade (in comparison with less than two months
in mice). Moreover, given that both stochastic and quality control processes are thought to lead tothe death (atresia) of the vast majority of oocytes in vivo, it will be a challenge to determine how to
select those that are normal. However, the ability to derive oocytes in vitro would open up several
possibilities for research and even treatment, including:(i) By following the development of oocytes from iPS cells derived from patients heteroplasmic for
mtDNA, more could be learned about the bottleneck and how this can lead to significant increases
in the proportion of mutant mtDNA such that oocytes can even become homoplasmic, and how
this contrasts with purifying selection that is also thought to occur in the germ line. There are alsoquestions about mechanisms underlying such shifts, which could involve the loss or gain of cellswith poorly performing mitochondria, mitophagy to remove abnormal mitochondria, and/or
differences in rates of mtDNA proliferation and ratchet effects, that could favour mutant or normal
mtDNA27,85. All of these parameters may be influenced by the environment of the cells (availabilityof nutrients, oxygen, etc), which can be manipulated in culture in ways that are difficult in vivo.
(ii) By fusing cytoplasts from cells with one mtDNA haplotype with iPS cells carrying another, it
would be possible to create heteroplasmy deliberately with a range of haplotype combinations totest when and how reversion occurs and whether oocytes are prone or resistant to this.
(iii) iPS cells from the patient could be used to derive oocytes, which could be used for MST withoocytes from normal donors providing the cytoplasts. In cases of heteroplasmy, because the iPS
cells would be derived from somatic cells of the patient they are likely to have relatively low levels
84 Hikabe O, Hamazaki N, Nagamatsu G, Obata Y, Hirao Y, Hamada N, Shimamoto S, Imamura T, Nakashima K, Saitou M,

Hayashi K. Reconstitution in vitro of the entire cycle of the mouse female germ line. Nature. 2016 Oct 17.
85 Malena A, Pantic B, Borgia D, Sgarbi G, Solaini G, Holt IJ, Spinazzola A, Perissinotto E, Sandri M, Baracca A, Vergani L.
Mitochondrial quality control: Cell-type-dependent responses to pathological mutant mitochondrial DNA. Autophagy. 2016 Nov.

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Human Fertilisation and Embryology Authorityof the mutant mtDNA, even if her oocytes consistently have very high levels or are homoplasmic. It
may even be possible to select iPS cell clones from the patient with very low levels of mutant
mtDNA. This might even remove the need to carry out MST; although if the quality of thecytoplasm of in vitro-derived oocytes were low, providing new cytoplasm from a normal donor
oocyte would be beneficial.
(iv) If in vitro-derived oocytes have high quality cytoplasm, but poor nuclear DNA, then it may bepossible to derive oocytes from the patient via iPS cells with very low proportions of mutant
mtDNA, and then use these to provide autologous cytoplasts for MST using karyoplasts from thepatients naturally produced oocytes. (v) If patients have reduced fertility as suggested in some ofthe evidence we received (although this was at odds with the data from the use of PGD), then theability to derive oocytes in vitro may overcome this problem.

There is no evidence in all the studies so far that reversion begins prior to late blastocyst stages,
yet it can begin in relatively early passages of ES cells derived from such blastocysts. mtDNA
replication must occur in ES cells (even though they have very low levels of OXPHOS), but there
is currently little understanding of how and when mtDNA replication resumes in the early humanembryo and whether it is similar in the various cell types (epiblast and extraembryonic). There isalso evidence that replication of normal and mutant mtDNA differs in distinct cell types84, but notwhen this arises during development. Very recently it has been demonstrated that it is possible toculture human embryos from late blastocysts, at about 7 days post fertilisation, in a system thatmimics aspects of implantation and allows their development in three dimensions up to around 13
days86,87. It should now be possible to use such cultures to explore mtDNA dynamics andmitochondrial function during this critical period when extra-embryonic tissues begin to elaborate
and the epiblast forms the embryonic disc, just prior to gastrulation. In as much as these systems
faithfully recapitulate this early developmental phase, they might also address whether mtDNA
haplotype reversion occurs after MST or PNT. Although it may be feasible to modify the culturesto permit development through and beyond gastrulation, this would contravene the current legal
limit of 14 days. However, isolated regions could, in principle, be maintained for longer, to explore
what happens in cell populations that give rise to the germ layers and to primordial germ cells.

The panel suggests that research in the areas below may eliminate the risks of carryover orreversion and may be useful for future refinements of the techniques.
There are essentially two categories of options that might be used to reduce or potentiallyeliminate reversion to karyoplast-derived mtDNA, if this turns out to be a concern following clinicaluse of MST and/or PNT. One does not require knowledge of the underlying mechanism, butwould involve methods to reduce or eliminate carryover of karyoplast-associated mtDNA. The
other is predicated on at least some knowledge of the mechanism and is based on subsequent
management or manipulation of this to prevent reversion. It seems likely that the mechanism isdue either to some feature of the methodology that is common to MST and PNT (and SCNT), or
86 Deglincerti A, Croft GF, Pietila LN, Zernicka-Goetz M, Siggia ED, Brivanlou AH. Self-organization of the in vitro attached humanembryo. Nature. 2016 May 4.
87 Shahbazi MN, Jedrusik A, Vuoristo S, Recher G, Hupalowska A, Bolton V, Fogarty NM, Campbell A, Devito LG, Ilic D, Khalaf
Y. Self-organization of the human embryo in the absence of maternal tissues. Nature cell biology. 2016 May 4.

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to preferential replication of the karyoplast-derived mtDNA haplotype. The latter could be aconsequence of specific differences in the sequence of its D-loop, compared to that in thecytoplast (donor)-derived mtDNA haplotype. Notably, all these options should reduce or eliminate
normal as well as abnormal mtDNA if both are present within the karyoplast and lead towards
homoplasmy for the cytoplast (donor)-derived mtDNA haplotype. However, the safety and efficacyof these proposed methods will need exploration.

It may be possible to refine methods of micromanipulation when removing the karyoplast
containing the spindle or pronuclei in order to reduce the volume of the cytoplasm containing
mitochondria. This has already been done to some extent by the Newcastle Group using thepipette to pinch off excess cytoplasm as the karyoplast is being introduced into the recipient
zygote (Hyslop et al., 2016 and Professor Mary Herbert, unpublished evidence presented to thepanel). However, it would be challenging to avoid any transfer of mitochondria at all, and there isthe concern that even very low levels of mtDNA with a replicative advantage could eventually
predominate.

Whilst acknowledging that they are currently unlawful in the UK, the panel continues to support
the investigation of polar body transfer (PBT) methodologies4. The very low numbers of
mitochondria found particularly in in the 1st, but also the 2nd polar bodies make them ideal, readymade karyoplasts for transfer to a donor egg or zygote. In conjunction with the carry-overelimination approaches discussed above and below, they offer great potential for near eliminationof karyoplast-derived mtDNA. Following a detailed study in mice88, a recent publication described
the successful generation of human blastocysts following polar body transfer and showed that thelevels of aneuploidy were similar between PBT and control blastocysts89, underlining the promiseof this approach for treatment of women at risk of passing on mitochondrial disorders.

Mitophagy is the natural process by which cells eliminate damaged or unwanted mitochondria. It
falls within the general term of autophagy, which is a regulated process by which cells eliminate
part of their cytoplasm to cope with stress or damage, from which they can then recover. The
mechanisms make use of pathways within cells to eliminate proteins, macromolecular structures,or organelles, where these are marked by ubiquitination and broken down by proteasomes ordestroyed after fusion with lysosomes. There are at least two types of mechanism leading tomitophagy:(i) The mechanism that operates in most cells, and which is often triggered by misfolded proteins
88 Wang T, Sha H, Ji D, Zhang HL, Chen D, Cao Y, Zhu J. Polar body genome transfer for preventing the transmission of

inherited mitochondrial diseases. Cell. 2014 Jun 19;157(7):1591-604.
89 Ma H, O’Neil RC, Gutierrez NM, Hariharan M, Zhang ZZ, He Y, Cinnioglu C, Kayali R, Kang E, Lee Y, Hayama T. Functional
Human Oocytes Generated by Transfer of Polar Body Genomes. Cell Stem Cell. 2016 Nov 10. In Press.

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inside the mitochondrial compartment, involves the proteins PINK1 and Parkin, which label
dysfunctional mitochondria for degradation90,91,92,93. Mitochondrial fission contributes to thisprocess by separating damaged segments of mitochondria from healthy ones94. The use ofagents to disrupt protein folding within mitochondria, or target misfolded proteins, or PINK1 andParkin, or other triggers of mitophagy of the mitochondria specifically in the karyoplast (or in theoocyte or zygote prior to its removal), might lead to the specific destruction of the karyoplastderived mitochondria in the early embryo. Recent data suggest that pp242, an ATP-competitivemTORC1/mTORC2 kinase inhibitor, can trigger mitophagy, at least in tumor cells95. This, orsimilar small molecules, could be used if shown to be safe and their action can be restricted to theelimination of karyoplast-derived mitochondria. Diot et al (2016) suggest that autophagy (including
mitophagy) occurs in early cleavage stage mouse embryos and that mitochondria removed by thisprocess may tend to be those with the highest levels of mutant mtDNA and the least capacity foroxidative phosphorylation. They also suggest that two known activators of mitophagy, rapamycin
and phenanthroline, can function in two-cell stage mouse embryos. Perhaps they would also workin oocytes or zygotes.
(ii) The other mechanism of mitophagy is that used to eliminate the mitochondria that are present
in the sperm after fertilisation. There are two reasons proposed for why this happens naturally: toprevent heteroplasmy in the resulting embryo, and/or to eliminate mtDNA that is likely to bedamaged by high levels of ROS in the sperm96,97. The mechanism relies on ubiquitination of
mitochondrial membrane proteins within the sperm, although the details of how and exactly when
this occurs is not well understood. After fertilisation, however, recent evidence suggests thatmitophagy of sperm mitochondria in the zygotes of higher mammals relies on a combined actionof canonical ubiquitin-like autophagy receptors, notably sequestosome 1 (SQSTM1), and valosincontaining protein (VCP)-mediated dislocation and presentation of ubiquitinated sperm
mitochondrial proteins to the 26S proteasome. This may explain how all the sperm mitochondria
are degraded inside fertilised oocytes by a protein recycling system usually involved indegradation of single protein molecules. If the mechanism for ubiquitinating the mitochondria insperm could be hijacked and repurposed to specifically ubiquitinate those in the karyoplast (or in
the oocyte or zygote prior to its removal), then this should automatically lead to their degradationin the early embryo in an analogous manner.
It may be possible to harness or modify these natural processes of mitophagy to eliminate
karyoplast-derived mitochondria prior to MST or PNT. Following refinement in cell lines andanimals, the safety and efficacy of these approaches could be assessed in human embryos.

90 Narendra D, Tanaka A, Suen DF, Youle RJ. Parkin is recruited selectively to impaired mitochondria and promotes theirautophagy. The Journal of cell biology. 2008 Dec 1;183(5):795-803.
91 Pickrell AM, Youle RJ. The roles of PINK1, parkin, and mitochondrial fidelity in Parkinson’s disease. Neuron. 2015 Jan
21;85(2):257-73.
92 Pellegrino MW, Haynes CM. Mitophagy and the mitochondrial unfolded protein response in neurodegeneration and bacterial
infection. BMC biology. 2015 Apr 3;13(1):1.
93 Haroon S, Vermulst M. Linking mitochondrial dynamics to mitochondrial protein quality control. Current opinion in genetics &development. 2016 Jun 30;38:68-74.
94 Twig G, Elorza A, Molina AJ, Mohamed H, Wikstrom JD, Walzer G, Stiles L, Haigh SE, Katz S, Las G, Alroy J. Fission andselective fusion govern mitochondrial segregation and elimination by autophagy. The EMBO journal. 2008 Jan 23;27(2):433-46.
95 Gordeev SA, Bykova TV, Zubova SG, Bystrova OA, Martynova MG, Pospelov VA, Pospelova TV. mTOR kinase inhibitor pp242
causes mitophagy terminated by apoptotic cell death in E1A-Ras transformed cells. Oncotarget. 2015 Dec 29;6(42):44905.
96 Song WH, Yi YJ, Sutovsky M, Meyers S, Sutovsky P. Autophagy and ubiquitin–proteasome system contribute to sperm
mitophagy after mammalian fertilization. Proceedings of the National Academy of Sciences. 2016 Sep 6;113(36):E5261-70.
97 Song WH, Ballard JW, Yi YJ, Sutovsky P. Regulation of mitochondrial genome inheritance by autophagy and ubiquitinproteasome system: implications for health, fitness, and fertility. BioMed research international. 2014 Jun 17;2014.

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Experiments on cells in culture and in mice suggest that specific restriction enzymes, zinc finger
nucleases (ZFN) and TALE nucleases (TALENs), each of which can be designed and constructedin the lab, can be targeted to mitochondria (using one of the peptide sequences involved in thenormal transport of nuclear-encoded proteins to mitochondria) in order to make a double-stranded
break in specific sequences within mtDNA leading to its degradation98,99,100,101,102. Notably,
TALENs have been used to eliminate a specified mtDNA haplotype in heteroplasmic 1-cell mouseembryos, which on transfer can give healthy mice that are homoplasmic for the mtDNA haplotype
that was not targeted100. Professor Ispizua-Belmonte submitted evidence to the panel, in which hediscussed these methods and their possible uses. These approaches were originally developedwith the idea of eliminating mutant mtDNA from early embryos or mature tissues in cases ofheteroplasmy.
However, it was recognised they would be inappropriate for cases of homoplasmy, or where thelevel of heteroplasmy for the abnormal mtDNA was very high, because they would reduce mtDNAlevels below those required for viability100. One potential way to overcome this problem, assuming
there was at least a proportion of normal mtDNA, would be to introduce mtDNA replication factors(that are normally absent in the early embryo) at the same time as the targeted nuclease. Thisshould promote several rounds of replication of the normal mtDNA, while the mutant version is
being destroyed; but this approach is untested and may be difficult to control.
One other complication is that the method originally proposed and tested in mice requires
targeting the specific sequence that is mutated in the abnormal mtDNA while leaving the normal
version untouched. This will depend on the specific mutation and may be technically challenging.
Nevertheless, these nuclease-based methods might be an ideal way to eliminate carryover after
MST or PNT. This will require detailed knowledge of the mtDNA sequences in both the patient andthe oocyte donor in order to design, for example, a TALEN that will specifically cut the mtDNA
haplotype from the former. However, it does not need to distinguish the specific mutation, but only
one or more of the several variant sequences that will distinguish the two haplotypes, making iteasier to design.
There will be a concern that the nucleases could also affect “off-target” sequences within the
nuclear genome. If these were cut, then processes of DNA-repair, such as non-homology end
joining (NHEJ) would operate, with the likelihood that they will lead to a small insertion or deletion(INDEL), which could have deleterious consequences if it affected a protein coding region or
critical regulatory region of a gene. (N.B. the mechanisms of NHEJ and homology-directed repair(HDR), which both operate on double-stranded breaks in DNA within the nucleus, do not occur
within mitochondria.) It is therefore critical to establish that the targeting of the nucleases tomitochondria is highly efficient, and/or that the target sequence within the mtDNA is absent fromboth the patient’s nuclear genome and that of her partner. Again, the safety and efficacy of these98 Because there is no obvious means by which guide RNAs could be targeted to mitochondria, the CRISPR/Cas9 system iscurrently unlikely to be useful in these approaches and mtDNA repair doesn’t operate efficiently.
99 Gammage PA, Gaude E, Van Haute L, Rebelo-Guiomar P, Jackson CB, Rorbach J, Pekalski ML, Robinson AJ, Charpentier M,
Concordet JP, Frezza C. Near-complete elimination of mutant mtDNA by iterative or dynamic dose-controlled treatment with
mtZFNs. Nucleic Acids Research. 2016 Sep 19;44(16):7804-16.
100 Diot A, Dombi E, Lodge T, Liao C, Morten K, Carver J, Wells D, Child T, Johnston IG, Williams S, Poulton J. Modulating
mitochondrial quality in disease transmission: towards enabling mitochondrial DNA disease carriers to have healthy children.
101 Bacman SR, Williams SL, Pinto M, Peralta S, Moraes CT. Specific elimination of mutant mitochondrial genomes in patientderived cells by mitoTALENs. Nature medicine. 2013 Sep 1;19(9):1111-3.
102 Reddy P, Ocampo A, Suzuki K, Luo J, Bacman SR, Williams SL, Sugawara A, Okamura D, Tsunekawa Y, Wu J, Lam D.
Selective elimination of mitochondrial mutations in the germline by genome editing. Cell. 2015 Apr 23;161(3):459-69.

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approaches – including potential effects on the sequence of nuclear DNA - should be carefully
assessed in human embryos generated by MST/PNT.
Although these methods might raise ethical and regulatory concerns about the use of genome
editing techniques to make heritable changes, in the scenarios being discussed here, the goal is toeliminate the targeted mtDNA haplotype so that it cannot be inherited. Moreover, the methods
involve the introduction of proteins (or perhaps mRNAs encoding them) in the oocyte or zygote,such that they will be present only transiently.
Mitochondria are often moved around within cells and form strong associations with organelles,
including the nucleus. This is presumably to maintain a ready source of ATP for processes that
require energy. These associations, which involve microtubules and/or microfilaments, are often
dynamic103,104,105,106. For example, mitochondria are largely excluded from the 1st polar body andthe set of chromosomes it contains, while perhaps remaining more closely associated with the set
that remains within the oocyte107,108.
There is also evidence that they are closely linked with the pronuclei (perhaps to facilitate DNA
replication)93. Any physical link might be difficult to disrupt by simple mechanical means. While theremoval of the karyoplast from either the oocyte or zygote makes use of cytoskeletal inhibitors,
perhaps the amounts used are insufficient to break these links. The panel therefore sees value in
research aimed at identifying the molecular nature of the putative links, as a means of shedding
light on the biology of mitochondria in the oocyte and zygote, and to determine whether they canbe disrupted using agents like nocodazole (which disrupts microtubules), cytochalasin B or D and
Latrunculin A (that disrupt microfilaments) at levels that do not interfere with viability, including
meiotic and mitotic cell divisions.

Repeated fusion and fission events involving mitochondria promote the equal distribution of theircontents, which would include mtDNA variants within a cell. In the fully-grown oocyte and early
embryo, mitochondria tend to be small and round, suggesting that fusion is infrequent 93,109. If so,
this might lead to retention of karyoplast-derived mtDNA in the subset of mitochondria carriedover after MST or PNT, rather than to their re-distribution throughout all the mitochondria. If the103 Katayama M, Zhong Z, Lai L, Sutovsky P, Prather RS, Schatten H. Mitochondrial distribution and microtubule organization in

fertilized and cloned porcine embryos: implications for developmental potential. Developmental biology. 2006 Nov 1;299(1):20620.
104 Shoji S, Yoshida N, Amanai M, Ohgishi M, Fukui T, Fujimoto S, Nakano Y, Kajikawa E, Perry AC. Mammalian Emi2 mediates
cytostatic arrest and transduces the signal for meiotic exit via Cdc20. The EMBO journal. 2006 Feb 22;25(4):834-45.
105 Koutsopoulos OS, Laine D, Osellame L, Chudakov DM, Parton RG, Frazier AE, Ryan MT. Human Miltons associate withmitochondria and induce microtubule-dependent remodeling of mitochondrial networks. Biochimica et Biophysica Acta (BBA)Molecular Cell Research. 2010 May 31;1803(5):564-74.
106 Chaigne A, Campillo C, Voituriez R, Gov NS, Sykes C, Verlhac MH, Terret ME. F-actin mechanics control spindle centring in
the mouse zygote. Nature communications. 2016 Jan 4;7. Pages/volume?
107 Dalton CM, Carroll J. Biased inheritance of mitochondria during asymmetric cell division in the mouse oocyte. J Cell Sci. 2013
Jul 1;126(13):2955-64.
108 Wang T, Sha H, Ji D, Zhang HL, Chen D, Cao Y, Zhu J. Polar body genome transfer for preventing the transmission of
inherited mitochondrial diseases. Cell. 2014 Jun 19;157(7):1591-604.
109 Takahashi Y, Hashimoto S, Yamochi T, Goto H, Yamanaka M, Amo A, Matsumoto H, Inoue M, Ito K, Nakaoka Y, Suzuki N.
Dynamic changes in mitochondrial distribution in human oocytes during meiotic maturation. Journal of assisted reproduction andgenetics. 2016 Apr 27:1-0.

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karyoplast-derived mitochondria were to become distributed equally throughout the cytoplasm of
the zygote, then the two haplotypes would become mixed and remain at a low level once
fusion/fission events begin in the late blastocyst or around gastrulation (assuming no difference inthe onset of mtDNA replication or its rate). However, if the karyoplast-derived mitochondria tend
to remain clustered, this mixing would be inefficient. This is in fact what tends to occur, at least inmouse zygotes and 2-cell embryos104. There is then a possibility that the phenomenon ofreversion is due to the karyoplast-derived mitochondria remaining associated in a cluster, suchthat during cleavage divisions they tend to end up in one or a few blastomeres, which by chance
may be those that give rise to the epiblast. (The ICM is derived from a few blastomeres that first
become inside cells in the morula; the epiblast is derived from a subset of the ICM cells, and theembryo proper (or ES cells) arise from only from a subset of the epiblast cells.)
Mitochondria are often associated with the cytoskeleton, which is used to move them around fromone part of a cell to where they are needed most. The cytoskeleton is also likely to be involved intheir fusion and fission107. Although the oocytes and zygotes used in MST and PNT are treatedwith cytoskeletal inhibitors to allow removal or the spindle or pronuclei as karyoplasts, theinhibitors are not present in the medium when the karyoplasts are put in a drop of fusogen or when
the cytoplast plus karyoplasts are incubated to allow fusion and subsequent development. Thismeans that the karyoplast-derived mitochondria may well begin to re-cluster before associatingwith those in the cytoplast, and then the two populations may tend to remain separately clusteredduring subsequent cleavage divisions.
Using MitoTracker, or a fluorescent protein tagged so that it labels mitochondria, it should bepossible to follow distribution and segregation of karyoplast-derived mitochondria during cleavage
divisions110. If clustering and segregation into a subset of blastomeres is seen, then it might bepossible to disrupt this in the zygote by brief incubation in cytoskeletal inhibitors, or to at least to
include these along with the karyoplast with the fusogen, while being careful to avoid missegregation of chromosomes and aneuploidy.
Pronuclear stage zygotes seem fairly resistant to disruption of cytoplasmic components, given thatrabbit, sheep, cow and pig zygotes can withstand centrifugation in order to visualise the pronuclei
when making transgenic animals via microinjection110,111, 112. Indeed, centrifugation would
concentrate all mitochondria to one pole of the zygote and these would then become redistributed,
presumably at random throughout the cytoplasm113.

110 Yamochi T, Hashimoto S, Amo A, Goto H, Yamanaka M, Inoue M, Nakaoka Y, Morimoto Y. Mitochondrial dynamics and theirintracellular traffic in porcine oocytes. Zygote. 2016:1-2.
111 Tatham BG, Sathananthan AH, Dharmawardena V, Munesinghe DY, Lewis I, Trounson AO. Fertilization and early
embryology: Centrifugation of bovine oocytes for nuclear micromanipulation and sperm microinjection. Human reproduction. 1996
Jul 1;11(7):1499-503.
112 Hirabayashi M, Hirao M, Takahashi R, Kimura K, Hirasawa K, Ueda M, Hochi S. Production of transgenic rabbits using
centrifuged pronuclear zygotes. J Vet Med Sci. 2000 Oct;62(10):1047-52.
113 Ferreira CR1, Burgstaller JP, Perecin F, Garcia JM, Chiaratti MR, Méo SC, Müller M, Smith LC, Meirelles FV, Steinborn
R.Pronounced segregation of donor mitochondria introduced by bovine ooplasmic transfer to the female germ-line. Biol Reprod.
2010 Mar;82(3):563-71.

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Human Fertilisation and Embryology Authority

It may be possible to choose a compatible donor according to likely dominance over the patient
haplotype, thereby reducing the risk of reversion. Whilst Kang et al (2016) tested only a limited
number of combinations, they propose that there could be a hierarchy of replication advantage
amongst mtDNA haplotypes (H56>H1b, U5A>H1b>F1a, U5a>X2c>D4a and H49>B2k). Further
research is required to substantiate and expand such a hierarchy in order to determine its
applicability to donor selection. It would be necessary, first, to systematically explore which D-loop
sequences confer a replicative advantage over others in vitro by making cybrid cell lines where
cytoplasts carrying one mtDNA haplotype are fused with cells carrying another. This could evenbe done with iPS cells derived from patients and potential donors. Once sufficient knowledge isavailable, it might be possible to predict simply on the basis of the D-loop sequences which
combinations would favour the donor haplotype. However, it may also be necessary to makeallowance for variation in the nuclear-encoded mtDNA replication factors114, although this can also
be tested. It is also worth noting that effects mediated by mtDNA sequence variants are likely tooperate at the same time as stochastic or bottleneck effects.

As noted in Section 4.8, methylation of specific D-loop sequences has been linked with reduced
mtDNA copy number. Rather than targeting a nuclease to a specific DNA sequence, TALENs withan inactive or “dead” form of the Fok1 nuclease (dFok1) linked to a DNA methyltransferase
protein should result in DNA methylation around their target sequence115. If this approach can beused to target the D-loop sequences involved in mtDNA replication specifically of the karyoplastassociated haplotype, this should make them less likely to replicate, allowing the cytoplast mtDNA
haplotype to predominate. This could rely on differences in the D-loop sequences of the two
haplotypes, or by introducing the TALENs into the patient’s oocyte or zygote prior to MST or PNT.
However, this assumes that the methylation will persist until mtDNA replication begins.
Methylation-based disruption of mtDNA replication has the advantage of not introducing a DNAcutting enzyme into the cell, with attendant risks of unwanted cutting. Again, however, whether
such approaches would be efficacious, safe and lawful is unclear.

114 Akman G, Desai R, Bailey LJ, Yasukawa T, Dalla Rosa I, Durigon R, Holmes JB, Moss CF, Mennuni M, Houlden H, Crouch

RJ. Pathological ribonuclease H1 causes R-loop depletion and aberrant DNA segregation in mitochondria. Proceedings of the
National Academy of Sciences. 2016 Jul 8:201600537.
115 Liu XS, Wu H, Ji X, Stelzer Y, Wu X, Czauderna S, Shu J, Dadon D, Young RA, Jaenisch R. Editing DNA Methylation in the
Mammalian Genome. Cell. 2016 Sep 22;167(1):233-47.

Scientific review: 2016 update

Human Fertilisation and Embryology Authority

The purpose of this review is to evaluate, in light of new evidence, whether techniques to avoid
mitochondrial disease are sufficiently safe to be offered to patients in a clinical setting.
In order to carry out this review, the HFEA convened a small panel to collate and summarise the current
state of expert understanding on the efficacy and safety of pronuclear transfer and maternal spindle
transfer techniques. Panel members, some of whom sat on the panel which produced the 2011 and 2013,
and 2014 review, were selected for their broad-ranging scientific and clinical expertise, and for having no
direct interests in the outcome of the review.
Panel members for this review are as follows:

Dr Andy Greenfield (Chair), Medical Research Council (MRC) Harwell Institute and HFEA memberProfessor Peter Braude, King’s College London, UKProfessor Frances Flinter, Guy’s & St Thomas’ NHS Foundation Trust, UKProfessor Robin Lovell-Badge, Francis Crick Institute, UKProfessor Caroline Ogilvie, King’s College London and Guy’s & St Thomas’ NHS Foundation Trust, UKDr Tony Perry, University of Bath, UK.

The panel published a call for evidence on 13 July 2016. It asked for scientific evidence from experts in
any relevant field on the safety or efficacy of pronuclear transfer and maternal spindle transfer techniquesto avoid the transmission of mitochondrial disease, including published studies (which have not been
submitted in previous calls for evidence or have been published since January 2013), unpublishedresearch or statements from individuals or organisations.
The call for evidence was published on the HFEA website and sent directly to more than 60 professional
bodies and experts in the field, the majority of whom had been sent the call for evidence for the original
review. The deadline for submissions was 21 July 2016.
The panel then reviewed the submitted evidence. It also spoke to the following researchers and experts
for additional information and clarification, at meetings held on 18 and 19 July and 14 October:Professor Dieter Egli, Columbia University, USA

Professor Mary Herbert, Newcastle University, UKProfessor Shoukhrat Mitalipov, Oregon Health and Science University, USADr Sherman Silber, St Luke’s Hospital, St Louis, Missouri, USA

Professor Hubert Smeets, University of Maastrict, The Netherlands

Professor Douglas Turnbull, Newcastle University, UK

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Human Fertilisation and Embryology Authority

Dr John Zhang, New Hope Fertility Centre, New York, USA.

The panel contacted a number of researchers after the call for evidence, to gather further expertise andclarify issues. Annex C lists the written evidence that the panel reviewed.

Scientific review: 2016 update

Human Fertilisation and Embryology Authority

Date

Consideration

2005

Research licence for pronuclear transfer granted.

May 2010

The Authority’s Scientific and Clinical Advances Advisory Committee
considered research developments.

February 2011

The Secretary of State for Health asked the HFEA to carry out a scientificreview to scope “expert views on the effectiveness and safety of
mitochondrial transfer”.

April 2011

The panel of experts, co-ordinated by the HFEA, reported to the Secretary ofState for Health on the safety and efficacy of methods to avoid mitochondrial
disease.

June 2011

The Authority’s Ethics and Law Committee considered ethical issues.

January 2012

The Secretary of State for Health and the Secretary of State for Business,
Innovation and Skills asked the HFEA to carry out public dialogue work on
the ethics and public attitudes towards mitochondrial replacement.

January 2012 – August Public dialogue and consultation work planning and preparation. Public
2012
dialogue work took place (deliberative public workshops and public
representative survey took place).
September 2012 –December 2012

Open consultation ran (open consultation questionnaire, open consultation
meetings and patient focus group).

December 2012

The Secretary of State for Health asked the HFEA to provide an updated
view of the science to support the assessment of the efficacy and safety of
MST and PNT.

Scientific review: 2016 update

January 2013

Human Fertilisation and Embryology Authority

The panel of experts was reconvened and call for evidence issued.
Key findings include: The panel’s view remained as it was in 2011: that MST and PNT have thepotential to be used for all patients with mtDNA disorders, which may
make them preferential to PGD in the future. In patients with homoplasmy(all containing mutant mtDNA) or high levels of heteroplasmy (partiallycontaining mutant mtDNA), these are the only techniques that would makeit possible for them to have a genetically related unaffected child.

March 2013

The panel was of the view that there was more published work available
to support MST than PNT, but there was still insufficient evidence torecommend one transfer technique over the other.

Once assessed as safe to use in clinical practice, the panel strongly
recommended that permission should be sought from the parents of thechildren born from MST or PNT to allow them to be followed up for an
extensive period (and that permission should then be sought from thechildren themselves, when old enough). The panel recommended that any
female born following MST or PST is advised, should she wish to havechildren of her own, that her oocytes (eggs) or early embryos areanalysed by PGD in order to select for embryos free of abnormal mtDNA.
This has the potential to eliminate risk in subsequent generations.



The 2013 panel continued to recommend the set of minimum criticalexperiments first outlined in the 2011 report. However, they highlighted
that the recommended work in understanding MST using fertilised oocytes
and PNT using normally fertilised oocytes was underway and noted that
progress was good. The panel’s comments on the progress made on therecommended research are summarised in Annex C of the 2013 report.

Further studies on mosaicism in human morulae (comparing individual
blastomeres) and on human embryonic stem (ES) cells (and theirdifferentiated derivatives) derived from blastocysts, where the embryos
have (i) originated from oocytes heteroplasmic for mtDNA and (ii) beencreated through MST and PNT using oocytes or zygotes with two different
variants of mtDNA. Although experiments are already reported onembryonic stem (ES) cells and their derivatives with MST, further
corroborative experiments would be valuable.



A recommendation made by the panel in 2011 to carry out PNT in nonhuman primate models was considered, in the light of new evidence, to beboth difficult and unnecessary. Such experiments were therefore nolonger mandatory.

The findings of the public dialogue and the 2013 scientific review update
were submitted to Government, together with considerations of how thetechniques might be regulated. The public dialogue work concluded that the
public were generally supportive of these techniques, although concerns
around safety, the donor role and the regulation of the techniques werehighlighted.

Scientific review: 2016 update

Human Fertilisation and Embryology Authority

June 2013

The Government announced that, based on the findings of the HFEA’s publicdialogue and consultation exercise and the views of the panel, it would move
forward with draft regulations for public consultation.

February 2014

The Department of Health opened a consultation on draft regulations for theuse of these techniques to prevent mothers passing on serious mitochondrial
diseases to their children.

June 2014

Alongside the public consultation on draft regulations for the use of thesetechniques, the Secretary of State for Health asked the HFEA to carry out afurther scientific review to evaluate the latest evidence on the effectivenessand safety of mitochondrial transfer.

February 2015

Parliament approved the regulations that permitted the use of mitochondrial
donation, by PNT or MST.

October 2015

The Regulations on mitochondrial donation to avoid serious mitochondrial
disease officially came into force. The statutory provisions governing this newtreatment are set out in the HFEA licensing and regulatory framework andprescribe the steps UK clinics must take before they can offer this newtreatment.
In order to trigger the licensing process and enable clinics to apply to belicensed to offer the treatment, a scientific panel would need to bereconvened to assess whether the outstanding three tests have been met
and there is sufficient evidence to proceed.
The HFEA agreed a draft licensing framework that would be put in place if
the techniques were deemed sufficiently safe and efficacious.

Scientific review: 2016 update

Human Fertilisation and Embryology Authority



A statement identifying studies by Yamada et al. (2016) and Cagone et al. (2016). Professor J Poulton(University of Oxford, UK) and Professor J St John (Centre for Genetic Diseases, Clayton, Australia).



A statement expressing opinion of the findings of Yamada et al. (2016) and Hyslop et al. (2016).
(anonymous, confidential).



A statement responding to 2014 report focussing on comparator experiments for PNT and mtDNA
haplotyping. Professor D Thorburn (University of Melbourne and Murdoch Childrens ResearchInstitute, Australia).



A statement, with references, expressing concerns about the use of MST/PNT in treatment. Dr E Allan,UK.



A statement regarding alternative techniques to prevent germline transmission of mitochondrial
disease. Professor JC Izpisua Belmonte (Salk Institute for Biological Sciences, USA).



A preprint manuscript: Differential gene expression in blastocysts following pronuclear transfer, 2016.
Dr E Morrow and Dr F Ingleby (University of Sussex, UK).

A statement in response to PeerJ non-peer reviewed article from Morrow and Ingleby. Professor M
Herbert (University of Newcastle, UK), Dr P Blakeley and Dr K Niakan (The Francis Crick Institute,UK), (confidential).



A published manuscript: mtDNA diversity in human populations highlights the merit of haplotype
matching in gene therapies. Dr I Johnston (University of Birmingham, UK).



Unpublished manuscripts and data from Professor M Herbert and Professor Sir D Turnbull (Universityof Newcastle, UK), (confidential):– 1. mtDNA carryover in individual cells from cleavage stage embryos/ morulae.
– 2. Heteroplasmy in embryonic stem cell lines from ePNT blastocysts.
– 3. Analysis of variant mitochondrial genotypes in hESCs lines.

Unpublished manuscripts from Professor S Mitalipov (Oregon Health and Science University, USA),(confidential):– 1. Mitochondrial replacement in human oocytes carrying pathogenic mtDNA mutations.
– 2. Functional Haploid Human Oocytes Generated from Polar Body Genomes.



Unpublished manuscript: Mitochondrial DNA inheritance is determined by genetic drift and negativeand positive selection from Professor H Smeets (University of Maastrict, The Netherlands),(confidential).

Scientific review: 2016 update

Human Fertilisation and Embryology AuthorityAddendum to the 2014 update - Review of the safety and efficacy of polar body transfer to avoid
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Activate

To artificially trigger the processes that occur when an egg is fertilised.

Adenosine
triphosphate (ATP)Molecule which transports energy in cells. It is involved in reactions of theelectron transfer chain (ETC).

Apoptosis

Programmed cell death.

Autophagy

Where a cell begins to digest part of itself.

Backcross

Formally, breeding a hybrid animal with one of its parents. In this context,
where a hybrid breeds with one of its parental types, such as a particularstrain.

BiopsyProcedure of obtaining cells, eg, from an embryo, in order to test them.

Blastocyst

A 5 to 6 day-old embryo, with both an outer trophectoderm cell layer and an
inner cell mass.

Blastomere

A cell of an embryo prior to the blastocyst stage.

Chromosome

Structure in a nucleus that carries DNA.

Cytoplast

An oocyte where the nuclear material has been removed.

Cytoplasm

The jelly-like substance that fills a cell and contains the structures (organelles)
within it, including mitochondria.

De novo mutation

A spontaneous new mutation (as opposed to one that is inherited).

Displacement (D)loop

A highly variable, non-coding region of mtDNA required for the initiation of
replication and transcription.

Electron transfer
chain (ETC)

The chain of chemical reactions that produces energy in a mitochondrion.

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Embryonic stem (ES) Cells, derived from an embryo and cultured in a laboratory, that have thecells
potential to form all different cell/tissue types in an animal or human and canreplicate themselves indefinitely.
Epigenetic

Relating to or arising from non-genetic influences on gene expression. Can
result in inherited phenotypes through alteration of gene expression.

Gene expression

The process by which information from a gene is used in the synthesis of afunctional gene product (which may or may not contribute to a particular
phenotype).

Genome editing

A term that covers a collection of related techniques that aims to introduce
specific alterations to DNA by the use of programmable enzymes. Pre-eminent
amongst these at present is CRISPR-Cas9, though other techniques areavailable or in development. Edits may be subtle, affecting single
nucleotides, or large, resulting in the complete removal of particular regions ofDNA. These edits may be restricted to somatic tissues or, if affecting germ line
cells, be heritable.

Germ cell

Any cell that will give rise to sperm or egg cells.

Germ line

The sequence of cells that give rise to sperm or egg cells that will pass genetic
information on to a child.

Germ line mosaicism Also known as gonadal mosaicism, a condition in which the precursor (germline) cells to ova or spermatozoa are a mixture (mosaic) of two or moregenetically different cell lines. If the mosaicism is present in only a small
proportion of cells, then it may lead to the inheritance of a condition from aparent who is unaffected by it.
Heteroplasmy

Where two or more different mtDNA types coexist in a single cell, commonlyused (as in this report) where one type is abnormal and the other normal.

Homoplasmy

Where all the mitochondria in a cell contain the same mtDNA, which can either
be all abnormal or all normal.

Induced pluripotent
stem (iPS) cells

Adult cells that have been reprogrammed to act like embryonic stem cells.

Inner cell mass

The mass of cells contained within a blastocyst stage mammalian embryo thatwill give rise to all cell and tissue types of the fetus.

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Karyoplast

A structure made up of the nuclear genetic material, surrounded by a small
amount of cytoplasm and enclosed in a portion of the cell membrane, used in
the current context to transfer the spindle or pronuclei.

Karyotype

The number and appearance of chromosomes in a cell.

Lineage

Term used to describe cells with a common ancestor.

Maternal spindle
transfer (MST)Technique to transfer the nuclear genetic material from a woman’s egg into a
donated egg with its nuclear genetic material removed.

Meiotic division

Type of cell division necessary for sexual reproduction.

Metaphase II oocyte

Mature oocytes or unfertilised eggs (that have undergone the first meiotic cell
division).

Micromanipulation

Use of physical techniques to add or remove cellular components.

Microtubules

Structural components of the cytoplasm, contributing to its ‘skeleton’ andinvolved in many cellular processes including cell division.

Mitochondria

Small structures present in cells that produce the cell's energy (seeIntroduction).

Mitochondrial
bottleneck

The description of a mechanism to explain how during the development of theembryo the low number of mitochondria per cell, and the small number of cells
that go on to form each tissue type, can by chance produce different
proportions of heteroplasmy in tissues.

Mitochondria
carryover

The proportion of mitochondria that is carried over from the original egg or
zygote during MST or PNT.

Mitochondrial DNA(mtDNA)The genetic material contained within the mitochondria.

Mitochondrial
haplogroup

A group of similar mitochondrial DNA types as defined by DNA sequence.

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Mitophagy

Cellular components are commonly broken down and recycled in a process
known as autophagy. Mitophagy describes the controlled destruction of
mitochondria. Turnover of mitochondria, especially if damaged, is important for
maintaining cellular health.

MitoTracker

Fluorescent dye that specifically stains mitochondria and which can persist in
living or fixed cells.

Mosaicism

When cells within the same individual have a different genetic makeup.

Mutation

A permanent, heritable change in the DNA sequence.

OocyteAn egg.

Ooplasm

Cytoplasm contained in an egg.

Organelle

Small structure within a cell.

Oxidative
phosphorylation

The reaction that produces ATP to generate energy in the cell.

Parthenogenesis

The generation of embryos without fertilisation of an egg. Because eggs (andsperm) contain only half of the usual complement of human chromosomes,
parthenogenetic embryos will also be haploid. Haploid individuals are nonviable, but haploid cells may be cultured in vitro.

PhenotypeThe set of observable characteristics of an individual resulting from theinteraction of its genotype with the environment.

Preimplantation
genetic diagnosis(PGD)

A technique that removes and examines one or more cells from an early
embryo to identify those embryos that are unlikely to develop a specific genetic
disease.

Prenatal testing

Testing for diseases or conditions in a fetus before it is born.

Primordial germ cell

A specialised cell, formed at early stages of development, that can give rise toegg or sperm cells.

Pronuclei

The independent nuclei derived from the sperm and egg during the process of
fertilisation, as distinct from the nucleus formed from the combination of thematernal and paternal genomes that is present in every cell type from the 2cell stage onwards.

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Pronuclear transfer(PNT)

Technique to transfer male and female parental nuclear genetic material froma fertilised egg (zygote) into a donor zygote with its nuclear genetic material
removed.

Reagents

Chemicals used in experiments.

Segregation

The process that determines the genetic information present in different
tissues or organisms.

Selection

The process by which particular entities, such as genes, or cells, are favoured.

Sequencing

Method for determining the order (sequence) of chemicals that make up DNA
in an individual.

Sex selection

Selecting an embryo of a particular sex to avoid passing on a serious sexlinked condition.

Somatic cell

Any cell of an embryo or adult that is not a germ line cell.

Somatic cell nuclear
transfer (SCNT)

Fusion of an adult (somatic) cell with an egg that has had its nucleus removed.

SpindleA structure in a cell, made up of microtubules, that move the chromosomes
when the cell divides.

Stochastic

Describes an event that is unpredictable due to one or more random variables.

Transcription

The first part of the process of gene expression, whereby a complementaryRNA-copy of a sequence of DNA is made. This may then be translated tomake the protein encoded by the DNA.

Vitrify

To freeze (an egg or embryo) by plunging into liquid nitrogen.

ZygoteA fertilised egg.

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