Annual report 2008/09 - Human Fertilisation & Embryology Authority

ScientificHorizonScanningat the HFEAAnnual Report 2008/09

Contents31 Introduction 42 Outcomes of 2007/8 horizon scanning process 52.1 Trophectoderm biopsy 52.2 Use of tripronucleated (3PN) embryos 62.3 Microcytoplast cryopreservation of oocytes 82.4 Gene transfer into embryos 92.5 Gene transfer into male germ lines 112.6 Cryopreservation of immature testicular tissue and spermatogonial cells 122.7 Alternative methods of obtaining embryonic or embryonic-like stem cells 132.8 In vitro derived gametes 163 Issues identified in the 2008/9 horizon scanning process 184 References 29Human Fertilisation & Embryology AuthorityScientific Horizon Scanning at the HFEA

4Section 1IntroductionHorizon scanning is an early-warning system whichallows the HFEA to consider the legal, ethical andregulatory implications of any techniques thatresearchers or clinicians may wish to use in the futurein HFEA-licensed research or treatment. The Authoritycan then be prepared with information to make adecision on the potential licensing of techniques, orhave guidance in place to ensure that new treatmentsare carried out safely and appropriately. The HFEAcan also ensure that patients and the wider public aresuitably informed.This year the horizon scanning process has also beenused to gather information on techniques which theHuman Fertilisation and Embryology Act 2008 willpermit from October 2009 (eg, genetic modificationof embryos for research).Relevant research is identified throughout the yearby reviewing journals and attending conferences.Common themes are then identified and researchedfurther. Views are sought from experts, such asmembers of the HFEA’s Horizon Scanning Panel.This report summarises the HFEA’s horizon scanningactivity for 2008/9, and the outcomes from 2007/8,to inform interested researchers, clinicians, membersof the public and other stakeholders by outlining thehigh priority issues considered throughout 2008, anyresulting actions and the issues identified forconsideration in 2009. If centres wish to carry out anyof the techniques described in this report they wouldneed to inform the Authority or apply for a licenceand, where necessary, further consultation will becarried out. A licence committee of the Authoritywould consider the details of each individualapplication before the technique could be used.Human Fertilisation & Embryology AuthorityScientific Horizon Scanning at the HFEA

6Section 2Outcomes of 2007/8horizon scanning processThe HFEA’s view and outcomesThe HFEA’s Scientific and Clinical Advances AdvisoryCommittee (SCAAC) felt that fewer embryos will bebiopsied when trophectoderm biopsy is usedbecause fewer embryos would develop to day 5.They felt that it would be fairly easy for clinics to carryout this technique if they are already carrying outPGD. Clinics would just need to notify the HFEA ifthey wished to carry out this technique.The Committee thought that trophectoderm biopsyis safer than conventional embryo biopsy becausetaking cells from the trophectoderm is less likely toaffect the development of the embryo. TheCommittee discussed whether information fromtrophectoderm cells will be the same as that frominner cell mass cells and whether the results will beaffected by mosaicism. They decided that because alarger number of cells are extracted and analysedwhen trophectoderm biopsy is used, cell mosaicismwas not an issue. It was not thought that thetechnique would affect placenta development.The Committee recommended that there should beinformation for patients regarding trophectodermbiopsy on the HFEA website. Therefore patientinformation on preimplantation genetic diagnosis hasbeen updated accordingly. The HFEA Executive andLicence Committees will refer to the informationoutlined in the paper and minutes of the SCAACmeeting held on 21 May 2008 when receivingnotifications or licence applications from centreswhich wish to use this technique for treatment orresearch.2.2 Use of tripronucleated (3PN) embryosWhat is it?A normally-fertilised egg (zygote) has one set ofgenetic material from the egg and one from thesperm. Sometimes during IVF two sperm can fertilisean egg at the same time. Also, sperm with two setsof chromosomes (diploid), which form when there isan error in the process of sperm formation (a meioticerror), can fertilise an egg. This leads to the zygotecontaining a nucleus from the egg and two nucleifrom sperm. The nuclei at this stage are calledpronuclei and therefore the zygote is said to be‘tripronucleated’ (3PN). 3PN zygotes occur in about4-7% of IVF cycles and are automatically discardedfrom IVF treatment, as they would not lead to a viablepregnancy. If the extra paternal pronucleus isidentified and removed the embryos could potentiallybe used for research or treatment.What impact could it have?Tripronucleated embryos could potentially be correctedand used in IVF treatment cycles, instead of beingdiscarded, thereby increasing the number of embryosavailable for treatment. Embryo biopsy would have to becarried out before embryo transfer to check whetherthe correct pronucleus (ie, a paternal pronucleus) hadbeen removed.The technique could also be used in research togenerate embryonic stem cell lines from embryos thatwould otherwise have been discarded. 3PN zygotescould therefore potentially be an alternative source ofembryos for human embryonic stem cell research.What research has been carried out?Altering the number of sets of chromosomes andparental constitution of zygotes has been carried outefficiently in several mammalian species. Early studiesdemonstrated that a pronucleus can be removedfrom human embryos to create bi-pronucleateddiploid embryos, but the in vitro developmental abilityof these embryos was restricted 8 9 . There was alsouncertainty as to whether or not embryos inheritedchromosomes from both parents (heteroparental).Human Fertilisation & Embryology AuthorityScientific Horizon Scanning at the HFEA

Section 2Outcomes of 2007/8horizon scanning process7Recently, however, a Spanish group developed atechnique that improves the identification andremoval of the extra paternal pronucleus 10 . The groupmicrosurgically removed the pronucleus locatedfurthest from the second polar body in 3PN humanzygotes using cytoskeletal relaxing agents. Theresulting embryos were diploid and developed toblastocyst stage, with the majority being heteroparental.Use for treatmentThere has been one case report in Singapore ofcorrected 3PN human embryos being used forreproductive purposes, resulting in a live birth 11 .The child appeared developmentally normal.Use for researchOne US group recently used 3PN mice zygotes asrecipients in nuclear transfer 12 . Zygotes fertilised withtwo spermatozoa were generated and inhibitors wereused to temporarily block the cell during mitosis.All the chromosomes were then removed from thezygote and replaced with chromosomes from a donorembryonic stem cell. When the inhibitors wereremoved, development resumed and a blastocystformed. These blastocysts could potentially be usedto derive new embryonic stem cell lines.When could it be introduced in the UK?The technique has already been used for reproductivepurposes in other countries and improvements in thetechnique have recently been shown in human embryos.The potential for 3PN embryos to be used in stemcell derivation has been explored in a recent paper 12 .However the experiment described was conducted inmice and has not yet been transferred to humans.3PN embryos have been used for other researchpurposes and for example in the UK, the HFEA haslicensed one research project involving polypronucleatedembryos (research project R0026 atwww.hfea.gov.uk).All UK groups wanting to use 3PN embryos for stemcell research will require a research licence from the HFEA.The HFEA’s view and outcomesThe HFEA’s Scientific and Clinical Advances AdvisoryCommittee (SCAAC) discussed the technique. TheCommittee felt that it was very unlikely that clinics inthe UK would want to use corrected 3PN embryosfor treatment purposes; one case study is notsufficient to demonstrate that the technique could beused for treatment. The Committee thought thatresearchers would want to use 3PN embryos forstem cell research and that there are other researchapplications for 3PN embryos, for example looking atthe origin of hydatidiform moles.The Department of Health advised the HFEA thatthey would need to consider whether removing apronucleus from a zygote would be classed asaltering the nuclear DNA of an embryo and thereforewhether use of corrected 3PN embryos for treatmentwould be prohibited by the Human Fertilisation andEmbryology Act 1990 (as amended).Information about the use of 3PN embryos to createstem cells has been included in a literature reviewregarding alternative methods to derive stem cells(outlined in papers of 21 February 2008 SCAACmeeting), which the HFEA’s Research LicenceCommittee refers to when deciding whether creationand use of embryos for proposed research isnecessary or desirable.Human Fertilisation & Embryology AuthorityScientific Horizon Scanning at the HFEA

8Section 2Outcomes of 2007/8horizon scanning process2.3 Microcytoplast cryopreservationof oocytesWhat is it?Microcytoplast cryopreservation involves removing asegment of an egg (microcytoplast) and freezing it.These segments are then thawed and reconstructedinto a complete egg.What impact could it have?This technique could impact upon assistedreproduction if it provides a more effective way tocryopreserve eggs than conventional methods. Intheory, this technique could increase the survival rateof cryopreserved eggs as it increases the surfacearea to volume ratio of the egg.Survival of the total fragments (parent segments andmicrocytoplasts) was significantly higher (75.5%) thanthe survival of control whole eggs (64.2%) afterthawing. However the survival of parent eggsegments only (66.9%) was not significantly higherthan the control eggs.Electrofusion of frozen-thawed microcytoplastsegments with parent or recipient eggs was moresuccessful when carried out within the zona pellucida(91.4%), compared to those fused without zonapellucida (56.2%). The spindle was intact followingreconstruction of the segment and parent egg, butthe group did not investigate if the egg woulddevelop any further.Freezing egg segments would also allow ooplasm(cytoplasm) to be stored and potentially transferredto eggs or embryos in order to improve theirdevelopmental potential in assisted reproductiontreatment. This would not be permitted under theHuman Fertilisation and Embryology Act 1990 (asamended) because it would be classed as alteringthe mitochondrial DNA of the egg, which isprohibited. However, the Act contains a regulationmaking power to allow for eggs or embryos, whichhave had their nuclear or mitochondrial DNA altered,to be permitted for treatment designed to prevent thetransmissionof serious mitochondrial disease.What research has been carried out?The technique of microcytoplast cryopreservatonhas been developed in mice by a group in the US 14 .Eggs were micromanipulated to remove ooplastfragments (microcytoplasts). The microcytoplastswere then injected into the perivitelline space andcryopreserved along with the eggs they werederived from (parent eggs) and control eggs usinga conventional slow freezing technique. Themicrocytoplasts were thawed and used toreconstruct an egg by electrofusion, either withor without a zona pellucida.Human Fertilisation & Embryology AuthorityScientific Horizon Scanning at the HFEA

Section 2Outcomes of 2007/8horizon scanning process9When could it be introduced in the UK?The technique is at an early stage of developmentand has only been carried out in mouse eggsreported in one study. The technique has not yetbeen shown to be effective at producing eggs thatcan undergo fertilisation and develop.The HFEA’s view and outcomesThe HFEA’s Scientific and Clinical Advances AdvisoryCommittee (SCAAC) considered this technique.They advised that microcytoplast cryopreservationwas not a viable technique for treatment, but groupsmay want to use it for research. They thought themouse model was not relevant to human eggs andthat the safety issues of this technique have not yetbeen addressed. They expressed concerns that thetechnique would disrupt the internal components ofthe egg and there is a risk that the egg would bedestroyed. They pointed out that vitrification of eggscan offer survival rates of 96%, so there would notbe a demand for this technique.There are safety implications of using this techniqueas a means for ooplasm transfer. A recent study 15found significant physiological differences betweenheteroplasmic and control mice. Problems inheteroplasmic mice included pulmonary hypertension,increased body mass and fat mass and abnormalelectrolyte levels. The Committee suggested that,as ooplasm transfer appears to be significantlyassociated with the mitochondrial heteroplasmy,if children were to be conceived through ooplasmtransfer would need to be closely monitored forhealth problems.The HFEA Executive and Licence Committees willrefer to the information outlined in the paper andminutes of the SCAAC meeting held on 21 February2008 in the potential event of receiving notificationsor licence applications from centres which wish touse this technique in research.2.4 Gene transfer into embryosWhat is it?It is possible to introduce transgenes into humanembryos in order to create genetically modifiedembryos for research. This has been achieved withthe use of viral vectors, which is the most commonmethod of genetic modification.What impact could it have?The technique could be used to generategenetically modified embryonic stem (ES) cells forstudying human embryogenesis and modellingdisease. Investigating gene function in earlyembryogenesis could lead to the development ofmore objective criteria for embryo selection. Thetechnique could also be used to introduce a geneto increase the efficiency of stem cell derivation.What research has been carried out?One group has reported that transgenes can beintroduced into mouse and human embryos by viraltransgenesis (with lentiviral vectors), leading to theproduction of transgenic blastocysts. Stablytransfected ES cell lines were derived from themouse transgenic blastocysts 16 .There has been recent work on new efficientmethods to create transgenic rats. The “sleepingbeauty” transposon system has been found to bean efficient method to generate mutant rats in vivo.This method has been used to create bigenicmales in which the DNA becomes incorporatedin the germ cells 17 .There is evidence that lentiviral vectors are apromising new tool for gene transfer in mammals.These viruses have a long incubation period, they canefficiently incorporate a significant amount of geneticinformation into the DNA of the host cell and they canbe used in different species 18 19 20 21 22 . In particular ithas been suggested that they could be used tocreate genetic models in rats.Human Fertilisation & Embryology AuthorityScientific Horizon Scanning at the HFEA

10Section 2Outcomes of 2007/8horizon scanning processOne group has demonstrated that the trophectodermof mouse embryos can be infected with lentiviruswithout the inner cell mass becoming infected 20 . Thetrophectoderm acts as a barrier to the virus particlesand shields the inner cell mass cells from viralinfection (and therefore from genetic alteration). Thissuggests that lineage-specific genetic alteration ofembryos is possible.When could it be introduced in the UK?It has already been demonstrated that it is technicallypossible to produce transgenic human blastocysts.The Human Fertilisation and Embryology Act 1990 (asamended) will allow the HFEA to issue licences forthe creation of genetically altered embryos forresearch purposes from October 2009.The HFEA’s views and outcomesThe HFEA’s Scientific and Clinical Advances AdvisoryCommittee advised the Authority that it is likely theywould receive applications for research projectsinvolving genetic modification of embryos fromOctober 2009.The Committee considered that the reasons forconducting research into gene transfer into embryosare numerous. Examples of the kinds of research are:• research into human embryo development,specifically the roles of genes and growthfactors involved in early development• the development of more objective criteria forembryo selection, by investigating gene functionin early embryogenesis• research into the genetic background ofadverse medical conditions – geneticallymodified ES cells could be created to modelmedical conditions• research into the fate of cells during embryodevelopment• introducing a gene to increase the efficiency ofstem cell derivation.The potential benefits of this research include theincreased understanding of medical conditions, andmore effective stem cell derivation techniquesresulting in less wastage of embryos.The Committee highlighted the possibility ofmutations arising as a result of some of thetechniques, but felt that this would be less of an issuein research than treatment. They felt that the mostefficient method for gene transfer was the “sleepingbeauty” vector, which had been used in chickens andrats. This is integrated largely at random so there is arisk of inactivating genes, leading to mutations.The HFEA’s Ethics and Law Advisory Committeeconsidered the ethical and public interest issuesassociated with the genetic modification of embryosfor research. The Committee agreed that the publicwould benefit from an increased understanding of thekinds of research projects which might involve theuse of genetically modified embryos.The HFEA’s Horizon Scanning Panel discussed thepotential use of genetic modification at its meetings inJuly and November 2008. The Panel thought that thetechnique could be used to answer basic biologicalquestions about early human development and couldbe used in comparative studies of early development.For example, it could be used to compare humanand mouse embryo development, such as why thereare abnormal nuclei in human blastomeres.The Panel discussed how inserting genes instead ofRNA would genetically alter the embryos for a longerperiod. This would be useful if, for example, theembryos were going to be used to derive stem cells.The Panel felt that consideration would need to begiven to whether the research could be done onembryos of other species before it is applied tohuman embryo research.Human Fertilisation & Embryology AuthorityScientific Horizon Scanning at the HFEA

Section 2Outcomes of 2007/8horizon scanning process11A paper outlining current research in the field ofgenetic alteration of embryos, the potential methodsresearchers may wish to use for genetically alteringembryos and the views of the HFEA committees onthis topic is being produced. This will ensure theHFEA’s Research Licence Committee is sufficientlyinformed ahead of licence applications for thisresearch which the Authority could potentiallyreceive from October 2009.2.5 Gene transfer into male germ linesWhat is it?It is possible to transfer genes into stem cells thatwill form sperm (spermatogonial cells) by adenovirusvectors and transplant them into seminiferous tubules.What impact could it have?This technique could be used to introduce agerm cell growth factor into Sertoli cells, therebyrestoring spermatogenesis in infertile males.Resulting offspring would not show viral integration.It is thought that this is currently the only methodto restore spermatogenesis because of Sertolicell defects and further studies are necessary totest whether such stable infection occurs in themale germ line 23 .Human Fertilisation & Embryology AuthorityScientific Horizon Scanning at the HFEA

12Section 2Outcomes of 2007/8horizon scanning processWhat research has been carried out?Genes have been successfully introduced intothe chicken male germ line by transplantingretrovirus-infected spermatogonia into the testesof sterilised recipient cockerels 24 . The retrovirusinfectedcells restored spermatogenesis within9 weeks with approximately the same efficiencyas non-infected cells.In addition, adenoviruses can be used to integrategenes into the male germ cell line, a techniquewhich was successful with mouse spermatogonialcells 23 . The infected spermatogonial cells weretransplanted into the seminiferous tubules of infertilemice where they reinitiated spermatogenesis.However, the virus gene was not present in theoffspring, which suggests it was not stably integratedinto the germ line. This technique was also recentlyused in goats, where genes were stably integratedinto the germ cell lines which led to the productionof transgenic offspring 25 .When could it be introduced in the UK?To date the transplantation of transgenicspermatogonia to restore spermatogenesis hasbeen successful in the mouse and a group recentlyachieved success in the goat (the first largemammalian species). To date there is no evidenceof this technique being used in humans.The creation and transplantation of transgenichuman sperm to the testes of a man falls outsidethe remit of the HFEA. However, legislation prohibitssperm (which includes early sperm cells) being usedin HFEA-licensed treatment if its nuclear ormitochondrial DNA has been altered. Sertoli cells donot fall within the definition of ‘sperm’ in the HumanFertilisation and Embryology Act 1990 (as amended)therefore the legislation does not prevent the geneticalteration of Sertoli cells.The HFEA’s view and outcomesThe HFEA’s Scientific and Clinical Advances AdvisoryCommittee considered this technique and thoughtthat there were significant safety issues for genetransfer into male germ lines. It is likely to be a longtime before this technique is transferred to humans.2.6 Cryopreservation of immaturetesticular tissue and spermatogonial cellsWhat is it?Cancer treatment can often be toxic to the gonads.Men and boys producing mature sperm can preservetheir fertility by freezing sperm samples. However thisis not an option for pre-pubescent boys who do notproduce mature sperm. Recent studies have lookedinto freezing samples of pre-pubescent boys’immature testicular tissue containing stem cells thatwill form sperm (spermatogonial stem cells) and earlysperm cells (spermatogonia and spermatocytes).What impact could it have?The technique of cryopreserving immature testiculartissue and spermatogonial cells could be used topreserve fertility in pre-pubescent boys undergoingcancer treatment.Clinicians may be able to transplant this tissueback into the patient when they are older in thehope that this will allow them to produce spermnaturally. Alternatively, researchers may be able tomature sperm from this tissue in the laboratory, anapproach which would benefit patients sufferingfrom haematological malignancies that may bere-transmitted by transplantation. The spermcould then fertilise an egg in vitro and be used infertility treatment.What research has been carried out?An effective method for cryopreserving immaturehuman testicular sperm has been developed 26 .The method maintains spermatogonia, Sertoli cellsand the tissue structure during freezing, thawingand tissue culture. Spermatogonial stem cells,from cryopreserved immature non-human primatetesticular tissue, have been shown to be capableof beginning spermatogenesis 27 .Research has also investigated methods of testiculartissue transplantation. Freeze-thawed humancryptorchid testicular tissue has been grafted intomice for 21 days 28 . 14.5% of spermatogonia survived,with 32% of those showing proliferative activity.Human Fertilisation & Embryology AuthorityScientific Horizon Scanning at the HFEA

Section 2Outcomes of 2007/8horizon scanning process13Cryopreserved immature human testicular tissue hasalso been transplanted into mice for six months.Spermatogonia were able to survive and proliferateand a few spermatocytes and spermatid-like cellswere seen. However, normal spermatogenesis wasnot observed and there was an increasing loss ofspermatogonia over time 29 .In comparison, restoration of normalspermatogenesis after transplantation of mouseimmature testicular tissue has been achieved in mice,demonstrated by the production of mature spermand offspring 30 .When could it be introduced in the UK?Though groups have developed effectivecryopreservation protocols, they have not managedto restore normal spermatogenesis followingtransplantation.To date there is no evidence of in vitro spermatogenesisbeing carried out using immature human testiculartissue. Research into the use of human immaturetesticular tissue is still at a very early stage.The HFEA’s view and outcomesThe HFEA’s Scientific and Clinical Advances AdvisoryCommittee discussed this technique and concludedthat transplanting tissue back into the patient wouldhave a higher chance of success than in vitrospermatogenesis. The Committee thought that moreextensive studies in animals were needed to look intothe genetic and epigenetic issues raised by thetechnique. The Commitee also thought that thetimescale for in vitro spermatogenesis was likely to bequite long, maybe around 10 years, but that clinicswere likely to be interested in the technique.The HFEA and Human Tissue Authority have begundiscussions to ensure that this technique will beappropriately regulated after October 2009 when theHFE Act 1990 (as amended) extends the HFEA’sremit to include immature gametes.2.7 Alternative methods of obtainingembryonic or embryonic-like stem cellsWhat is it?There are a number of techniques being developed toderive embryonic stem (ES) cells or ES-like cells thatdo not rely on nuclear transfer into oocytes or destroyingviable embryos. These methods include:• Induced pluripotent stem cells (iPS) – Adultcells, such as skin cells, can be directlyreprogrammed into pluripotent cells. TheseiPS cells have similar properties to ES cells.• Embryonic stem cells from blastomeres –ES cells can be derived from a singleblastomere taken from an embryo. Theremaining embryo can then potentiallycontinue to develop as normal.• Parthenogenetic embryonic stem (pES) cells –Human pES cells can be generated throughartificial activation of oocytes to form diploidparthenotes (a process by which an egg candevelop into an embryo in the absence ofsperm). Parthenogenetic stem cell lines canthen be derived from these parthenotes.• ES cells from poor/non-viable embryos –Cells can be taken from poor quality or nonviableembryos and used to create ES cell lines.• Nuclear transfer into zygotes – Zygotes, asopposed to oocytes, can be used as recipientsof nuclear transfer. Stem cells can then bederived from the resulting embryos.• ES-like cells from other tissues – Populationsof stem cells, which express markerscharacteristic of ES cells populations, havebeen found in adult tissues and blood.What impact could it have?Paragraph 3(2) of Schedule 2 of Human Fertilisationand Embryology Act 1990 requires embryo researchto be “necessary or desirable” for defined purposes.If viable alternative methods of deriving ES or ES-likecells are developed a licence committee of theAuthority may judge that it is not “necessary” forresearch groups to destroy viable embryos.Human Fertilisation & Embryology AuthorityScientific Horizon Scanning at the HFEA

14Section 2Outcomes of 2007/8horizon scanning processIt is important for the Authority to keep up to datewith developments regarding these alternativemethods so the Research Licence Committee of theAuthority can bear them in mind when consideringlicence applications.What research has been carried out?Induced pluripotent stem cellsA combination of four factors, Oct3/4 and Sox2 witheither Klf4 and c-Myc or Nanog and Lin28, have beenshown to directly reprogram a range of adult human,mouse and monkey cells to form iPS cells 31 32 33 .The techniques used to introduce these factors intocells have traditionally involved the use of retrovirusesor lentiviruses. These can be mutagenic and potentiallyactivate oncogenes. Drug-inducible transgenic systemshave been developed to reprogram somatic cells intoiPS cells in the mouse and human 34 35 . Mouse iPScells have been generated without viral vectors usingrepeated transfection of two expression plasmids 36 .The potential use of iPS cells in gene or cell therapyhas been demonstrated 37 38 .Embryonic stem cells from blastomeresEmbryonic stem cell lines have been derived fromsingle mouse blastomeres, taken from 2, 4 and 8-cellembryos 39 . Human embryonic stem cell lines havebeen derived from a single blastomere taken from a4-cell stage embryo 40 and blastomeres removed from8-cell stage human embryos have been shown toform multi-cell aggregates 41 .There have also been reports of single embryosproducing both ES cell lines and offspring. An EScell line from a blastomere removed from a mouseembryo differentiated into some types of tissueand contributed to a chimera mouse, whilst theremaining embryo continued to develop andproduce offspring 42 . Another group reported thederivation of 5 human ES cell lines from individualblastomeres without destroying the embryo.The blastomeres were removed using a techniquesimilar to preimplanation genetic diagnosis 43 .A laser system has been developed to isolate theinner cell mass as an alternative to the usualimmunosurgery that utilises animal products 44 .Parthenogenetic embryonic stem (pES) cellsParthenogenetic embryonic stem (pES) cell linescontinue to be developed 45 46 47 48 and it has beenshown that pES cell lines can be derived from in vitromatured oocytes grown in preantral follicles 49 50 .Parthenogenetic embryonic stem cells, derived fromeggs, have been shown to exhibit ES cell pluripotencybut have poor differentiation potential in vivo 51 . Thedifferentiation potential of pES cells has been improvedby using a nuclear transfer technique whereby theoriginal pES cell nucleus is transferred into theenucleated oocytes and the resulting embryos establishnew nuclear transfer-pES cell lines 52 . It is suggestedthat this improvement could be due to epigeneticchanges brought about by nuclear transfer 53 .The pES cell genome carries entirely maternalimprinting. DNA that is normally paternally imprintedmay therefore be abnormally expressed 54 . Althoughthere is research that suggests not all pES cell linessuffer from this 55 , it is a significant limitation of pEScell application 48 .ES cells from poor/non-viable embryosWork has been published on a correlation betweenthe developmental stage of the embryo used andcreation of ES cell lines. Early-arrested or highlyfragmented embryos only rarely yield cell lines,whereas those that have achieved blastocyst stageare a robust source of normal hES cells 56 . A humanES cell line has also been generated from a singleblastomere of an arrested four cell stage embryo 57 .Nuclear transfer into zygotesA US group has reported the derivation of stemcells from such embryos. It also successfully usedtripronucleated (3PN) zygotes as recipients fornuclear transfer using the same technique, thoughdid not attempt to derive stem cells from theresulting blastocysts 58 .Human Fertilisation & Embryology AuthorityScientific Horizon Scanning at the HFEA

Section 2Outcomes of 2007/8horizon scanning process15Tripronucleated zygotes are automaticallydiscarded from IVF treatment. This techniquecould therefore potentially derive ES cells withoutdestroying viable embryos.HFEA Horizon Scanning Panel members, at ameeting in November 2008, thought that moreresearch was required into the degree of similaritybetween ES cells and iPS cells.ES-like cells from other tissuesPopulations of very small embryonic-like stem cellshave been derived from murine bone marrow andadult tissues 59 . Some of these cells express markerscharacteristic of ES cells and indications that theymay differentiate into all three germ layers 60 . There isevidence that the cells circulate at very low levels inperipheral blood and that similar ES-like cells existin human umbilical cord blood 61 . Some work hasbeen carried out looking at transplantation in mice 62 .Human adult germline stem cells have been derivedfrom spermatogonial cells of the adult human testis 63 .The human adult germline stem cells producedteratomas and differentiated into all three germ layerswhen grown under conditions used to induce thedifferentiation of human ES cells.The HFEA’s view and outcomesThe HFEA’s SCAAC considered a review of advancesin these techniques and concluded that whilstresearch into iPS cells and stem cells derived fromsingle blastomeres was progressing well, currently theonly feasible way to derive ES cells for therapy maybe from viable embryos.Information about the latest developments regardingthese alternative methods and the view of the HFEAgroups on this topic has been outlined in a paperwhich the HFEA’s Research Licence Committee refersto when deciding whether creation and use ofembryos for proposed research is necessary ordesirable (outlined in papers of 21 February 2008SCAAC meeting).ES cells from cloned embryosES cell lines have been produced from primatesomatic cell nuclear transfer (SCNT) embryos 64 andcloned human blastocysts have been producedfollowing SCNT with adult fibroblasts 65 . DNAfingerprinting analyses of some of the clonedblastocysts showed that the genomic DNA matchedthe donor fibroblast and was not of the oocyte or ofparthenogenetic origin. No attempt was made toderive ES cell lines from the cloned embryos.When could it be introduced in the UK?Members of the HFEA’s Scientific and ClinicalAdvances Advisory Committee (SCAAC) expressedthe view in 2008 that research into iPS cells and stemcells derived from single blastomeres was progressingvery rapidly. Both sources of ES or ES-like cells werethought to be more useful in research than therapyand given the legal prohibition, in the UK, of usingthe same embryo for both treatment and research,iPS cells appeared to be the most promisingalternative to ES cells.Human Fertilisation & Embryology AuthorityScientific Horizon Scanning at the HFEA

16Section 2Outcomes of 2007/8horizon scanning process2.8 In vitro derived gametesWhat is it?Stem cells have the potential to form differentcell types in the body. Potentially they can thereforebe used to form sperm and egg cells. Gametesformed in this way have been termed in vitroderived gametes.In vitro derived gametes can potentially be producedfrom a range of cells, including stem cells from bonemarrow, from embryos and from adult cells (such asskin cells) that have been reprogrammed to behavelike embryonic-like stem cells.What impact could it have?In vitro derived gametes could be used forresearching germ cell development, differentiation,meiosis and imprinting. They also have potentialclinical applications, such as treatment of infertilityand germline gene therapy.Haploid androgenotes (cloned sperm) couldpotentially be used where a man is producing lowlevels of sperm, or only a single sperm is extracted.This method could be used to propagate this spermand provide the opportunity to use replicates of thesperm nucleus for diagnostic purposes.In theory in vitro derived gametes could be used intreatment to allow men and women otherwise unableto produce gametes to have children geneticallyrelated to them. However, the Human Fertilisationand Embryology Act 2008, when it comes into forcein October 2009, will prohibit the use of gametesfor treatment which have not been produced orextracted from the ovaries of a woman or thetestes of a man.What research has been carried out?Bone marrow cellsSperm and oocyte germ cells have been derived frombone marrow derived mesenchymal stem cells (MSC) inthe mouse 66 67 . Bone marrow stem cells transplantedinto the testes of mice can differentiate into germcells lineages 68 . Early human sperm germ cells canalso be derived from bone marrow MSC, but thesesperm cells do not undergo meiosis 69 .Embryonic stem cellsES cells have been used to derive sperm germ cellsin both mice and humans 70 71 72 73 . In mice, the resultingsperm can give rise to offspring when injected intounfertilised eggs 74 . However the offspring wereabnormal due to defective imprinting and diedprematurely. Early oocyte-like germ cells have alsobeen derived from ES cells in mice 75 76 . One study hassuggested that both presumptive sperm and oocytescould be produced from XY ES cells in mice 77 . In2008 a group reported what they believed to be thefirst research showing evidence for in vitro productionof male gametes from ES cells without geneticmanipulation or pre-selection 78 .Human Fertilisation & Embryology AuthorityScientific Horizon Scanning at the HFEA

Section 2Outcomes of 2007/8horizon scanning process17The fusion of an embryonic stem cell with asomatic cell has been shown to reprogram thehybrid cell genome into one that allows differentiationinto primordial germ cells in vitro 79 . These cellscan then undergo further differentiation into germ-likecells and could provide personalised stem cellsapplicable to regenerative medicine and assistedreproductive technologies.Somatic cellsSomatic cells can be reprogrammed to behavelike ES cells. These induced pluripotent stem cells(iPS cells) could potentially be used to derivehuman sperm and eggs. In reviewing somatic cellhaploidisation, however, this technique presentsdifficulties in ensuring accurate chromosomesegregation and prevention of epigenetic defectsin imprinted genes of the somatic cell nucleus 80 .These limitations need to be overcome before thetechnique provides a valid method for derivinggametes in vitro.Haploid androgenotesA group from the US have reported that it is possibleto replicate a haploid male genome by injectingenucleated mouse eggs with a single healthy mousesperm. The sperm genome was found to bechromosomally identical to its originator in over 80%of the clones analysed. The resulting cells were fusedwith an egg that had previously been chemicallyactivated. The embryos derived had chromosomesfrom both parents, some of which resulted inoffspring. All of the offspring were phenotypicallynormal, some grew normally and some died soonafter birth 81 82 . This demonstrates that it is possible toreplicate the male genome and that such a clonedgenome has the ability to develop to term.When could it be introduced in the UK?So far researchers have not been able to derivemature human gametes from stem cells. However, inmice, in vitro derived sperm have been producedfrom stem cells capable of fertilising eggs andproducing offspring, although the offspring diedshortly after birth.Members of the Scientific and Clinical AdvancesAdvisory Committee (SCAAC) thought, in May 2008,that the timescale for deriving gametes for treatmentwas between 5-10 years. The timescale for derivingsperm from stem cells would be shorter (1-2 years)than that for deriving eggs.One member of the Horizon Scanning Panelthought, in November 2008, that within 12 monthsresearchers would wish to test the competency ofmale germ cells derived from human ES cells byinjecting them into human oocytes. Members believedthat such research must be at an appropriate stagefor using human material and reviewers must considerwhether the aim of the study could be achieved byalternative means.The one research group investigating haploidandrogenotes considers that the technique will not beused in humans in the near future. Further workneeds to be done to understand why impaireddevelopment and abnormalities in the embryo occur,and to take steps to avoid that occurrence.The HFEA’s view and outcomesThe HFEA Executive and Licence Committees willrefer to information and advice (outlined in the paperand minutes of the 21 May 2008 SCAAC meetingand the 7July and 6 November 2008 HorizonScanning Panel meetings) if they receive licenceapplications from centres wishing to use thistechnique for research.Human Fertilisation & Embryology AuthorityScientific Horizon Scanning at the HFEA

18Section 3Issues identified in the 2008/9horizon scanning processThis table presents all the issues that were identified through the 2008/9 horizon scanning process. It includesresearched published up to November 2008. Issues were prioritised using a systematic approach which looks atwhether:• the technique is transferable to humans for research or treatment,• the diffusion of the technique is likely to be rapid,• there will be public interest or concern,• there will be ethical or legal considerations, and• the technique is within the remit of the HFEA.High priority issues will be considered in depth by the HFEA’s Scientific and Clinical Advances Advisory Committeethroughout 2009. Low and medium priority issues will not be followed up in any detail but have been providedfor information.Embryo selectionIssue Priority Use or aim Description Research identifiedAssessment High To select Morphology of the early Hammoud I et al. (2008)of embryo embryos embryo can act as a marker Fertility and Sterility 90(3):551-556.morphology that will of embryo quality. Various Hesters L et al. (2008)improve ART methods of assessing Fertility and Sterility 89(6):1677-1684.outcomes. morphology have been Moayeri S et al. (2008)investigated including Fertility and Sterility 89(1):118-123.cleavage rate and Muna M et al. (2008)persistence of pronuclei. Fertility and Sterility 89(2):358-363.Metabolomic Medium To select The study of embryo culture Blow N. (2008) Nature 455:698.profiling of embryos media using techniques Scott R et al. (2008)embryo that will such as Raman or near- Fertility and Sterility 90(1):77-83.culture media improve ART infrared spectroscopy reveals Sel E et al. (2007)outcomes. a relationship between the Fertility and Sterility 88(5):1350-1357.metabolic profile of an Sturmey R et al. (2009)embryo and its quality. Human Reproduction 24:81-91.Vergouw C et al. (2008)Human Reproduction 23(7):1499-1504.Warner C et al. (2008)Reproductive BioMedicine Online17(4):470-485.A combination Medium To select Research into blastocyst Goossens V et al. (2008)of invasive embryos biopsy, microarray gene Human Reproduction 23(3):481-492.selection that will expression profiling and Jones G et al. (2008)strategies improve ART DNA finger printing as Human Reproduction 23(8):1748-1759.outcomes. methods of invasively Wang W-H et al. (2008)assessing embryo quality. Fertility and Sterility 90(2):438-442.Human Fertilisation & Embryology AuthorityScientific Horizon Scanning at the HFEA

Section 3Issues identified in the 2008/9horizon scanning process19Gamete selection, storage and manipulationIssue Priority Use or aim Description Research identifiedInterspecies High To select Interspecies fertilisation used Fulka H. (2008)fertilisation gametes to analyse sperm epigenetic Human Reproduction 23(3):627-634.that will status and normality.improve ARToutcomes.Gene Medium To select Identification, using Aafke P. et al. (2008)expression gametes microarrays, of gene Molecular Human Reproductionthat will expression patterns in single 14(3):157-168.improve ART embryo cells, polar bodies Cill F et al. (2007)outcomes. and cumulus cells can predict Reproduction 134:645-650.gamete quality. Feuerstein P et al. (2007)Human Reproduction 22(12):3069-3077.Hamel M et al. (2008)Human Reproduction 23(5):1118-1127.Jones G et al. (2008)Human Reproduction 23(5):1138-1144.Kearns W et al. (2008)Human Reproduction 23,Suppl:O-164, i66.Assessment Medium To select Levels of proteins, growth D’Aniello G et al. (2007)of factors gametes factors and cytokines in Human Reproduction 22(12):3178-3183.and proteins that will follicular fluid can predict Fujwara H and Matsubayashi Hin follicular improve ART oocyte and embryo quality. et al (2007) Human Reproductionfluid outcomes. 22(11):3042-3045.Ledee N et al. (2008).Human Reproduction 23,Suppl 1:O-063, i27.Ledee N et al. (2008)Human Reproduction 23(9):2001-2009.Lee J et al. (2008)Human Reproduction 23,Suppl 1:O-0179, i73.Sinclair K et al. (2008)Reproductive Biomedicine Online16(6):859–868.Takahashi C et al. (2008)Fertility and Sterility 89(3):586-591.Metabolomic Medium To select Metabolomic profiling of Behr B et al. (2008)profiling of gametes oocyte culture media to Human Reproduction 23,oocyte that will predict embryo development Suppl 1:O-205, i83.culture media improve ART and quality. Picton H et al. (2008)outcomes.Human Reproduction 23, Suppl 1:P-286.Human Fertilisation & Embryology AuthorityScientific Horizon Scanning at the HFEA

20Section 3Issues identified in the 2008/9horizon scanning processGamete selection, storage and manipulation (continued)Issue Priority Use or aim Description Research identifiedRespiration Medium To select Novel non-invasive method Scott L et al. (2008)rate as a gametes of assessing ability of oocytes Reproductive BioMedicine Onlinemarker of that will to respire at a rate that can 17(4):461-469.gamete improve ART sustain fertilisation andquality outcomes. development.Cumulus Medium To select Mixing sperm with cumulus Rijsdijk M & Franken D R (2007)oophorous gametes oophorous cells can help Fertility and Sterility 88(6):1595-1602.cell selection that will physiologically select spermimprove ART to be used in ICSI.outcomes.Assessment Medium To select Assessment of gamete Ebner T et al. (2008)of gamete gametes morphological characteristics, Reproductive Biomedicine Onlinemorphology that will such as smooth endoplasmic 16(1):113-118.improve ART reticulum aggregations in Gianaroli L et al. (2008)outcomes. oocytes, to assess quality Fertility and Sterility 90(1):104-112.eg, birefringence. Montag M et al. (2008)Reproductive BioMedicine Online17(4):454-460.Assessment Low To select Spindle visualisation in eggs Dubey A et al (2008)of gamete gametes predicts embryo development; Fertility and Sterility 89(6):1665-1669.morphology that will sperm morphology influences Madaschi C et al. (2008)improve ART rate of euploidy, implantation Fertility and Sterility 90(1):194-198.outcomes. and clinical pregnancy.DNA Low To select The level of DNA Collins J et al. (2008)fragmentation gametes fragmentation can predict Fertility and Sterility 89(4):823-831.that will sperm and embryo quality. Vagnin L et al. (2007)improve ARTReproductive Biomedicine Onlineoutcomes. 15(5):514-519.Automation Low To select Automation of aneuploidy/ Carrell D et al. (2008)of sperm gametes euploidy sperm sorting has Fertility and Sterility 90(2):434-437.selection that will similar accuracy to manualimprove ART sorting.outcomes.G6PDH Low To select G6PDH as a marker of Torner H et al. (2008)selection gametes bovine egg competency and Reproduction 135(2):197-212.marker that will underlying molecular andimprove ART subcellular differences.outcomes.Human Fertilisation & Embryology AuthorityScientific Horizon Scanning at the HFEA

Section 3Issues identified in the 2008/9horizon scanning process21In vitro derived gametesIssue Priority Use or aim Description Research identifiedGamete High Research In vitro production of Kerkis I et al. (2008)derivation into the germ-like cells and Human Reproduction 23,germ line. spermatogenesis. Suppl 1:O-228, i92.Lavagnolli T et al. (2008).Human Reproduction 23,Suppl 1:O-231, i94.Scholer H. (2008)Derivation of gametes from embryonicstem cells. Society for Reproduction andFertility conference.West F et al. (2008)Stem cells 26(11):2768-2776.Mature Medium Research Production of mature oocytes Qing T et al. (2008)oocyte into the from purified mouse fetal Human Reproduction 23(1):54-61.derivation germ line. germ cells.Oocyte Low Improve Gonadotrophins, steroids and Yeo C et al. (2008)Maturation outcomes of oocyte secreted factor GDF9 Human Reproduction 23(1):67-73.oocyte in vitro shown to increase embryo Murray A et al. (2008)maturation. development and fetal viability. Molecular Human Reproduction14(2):75-83.Germ line Low Research Discovered to be two distinct Izadyar F et al. (2008)stem cells into the populations of germ line Reproduction 135(7):177-784.germ line. stem cells, one gives rise tospermatogonial stem cellsand the other multipotent celllines with some pluripotentcharacteristics.Isolation of Low Research Comparison of two gradient Saiti D et al. (2008)primordial into the methods of isolation shows Reproductive BioMedicine Onlinegerm cells germ line. similar isolation rate but 16(5):730-740.from genitalmore germ cells with higherridgesviability using Percoll.Review of Low Research Mouse ES cells derived Wkayama T et al. (2008)technology into the from cloned and Reproductive BioMedicine Onlinegerm line. fertilised blastocysts are 16(4):545-552.transcriptionally andfunctionally indistinguishable.Human Fertilisation & Embryology AuthorityScientific Horizon Scanning at the HFEA

22Section 3Issues identified in the 2008/9horizon scanning processStem cell derivation and cloned embryosIssue Priority Use or aim Description Research identifiedHuman High Reduce need Pig ovaries may be a useful Braga D et al. (2007)admixed for human tool for oocyte retrieval and Fertility and Sterility 88(5):1408-1412.embryos oocytes for intracytoplasmic sperm Beyhan Z et al (2007)training injection. Cell Stem Cell 1(5):502-512.purposesand improvemethods ofnucleartransfer.Alternative High Alternative Induced pluripotent stem Aoi T et al. (2008)methods of methods of cells from a variety of cell Science 321(5889):699-702.obtaining deriving types, work on increasing Costello J (2008) Nature 454:45-46.embryonic embryonic efficiency of re-programming, Cyranoski D et al. (2008)stem cells stem cells or development of a drug Nature 452:132.or stem stem cell-like inducible system and Dimos J et al. (2008)cell-like cells cells without comparison of cell lines. Science 321(5893):1218-1221.destroying Hanna J et al (2007)viable Science 318:1920-1923.embryos. Hockemeyer D et al. (2008)Cell stem cell 3(3):346-353.Holden C et al. (2008)Science 319:560-563.Kim J et al. (2008)Nature 454:646-650.Maherali N et al. (2008)Cell Stem Cell 3(3):340-345.Mali P et al. (2008)Stem Cells 26(8):1998-2005.Park I-H et al. (2008)Nature 451(7174):141-146.Tada T et al. (2008)Cell stem cell 3(2):121-122.Takahashi et al (2007)Cell 131(5):861-872.Yu J et al. (2008)Genes and Development 22(15):1987-1997.Yu J et al (2007)Science 318:1917-1920.Human Fertilisation & Embryology AuthorityScientific Horizon Scanning at the HFEA

Section 3Issues identified in the 2008/9horizon scanning process25IVF/ICSI technologiesIssue Priority Use or aim Description Research identifiedDefective Medium Mitochondrial Suggestion that the ART Smith L. (2008)mtDNA disease techniques of cytoplasmic Mitochondrial DNA in the oocyte andreplacement treatment. and pronuclear transplantation embryo: consequences of ART.are effective means to replace Society for Reproduction and Fertilitydefective mitochondria in conference.maternal lineages.Ultrasound Low Improve ART Evidence for and against Ali R et al. (2008).guided outcomes. ultrasound guided embryo Reproductive BioMedicine Onlineembryo transfer improving 17(1):88-93.transfer pregnancy rate. Almodovar M et al. (2008).Human Reproduction 23,Suppl 1:O-55, i23.Drakeley A. et al. (2008).Human Reproduction 23(5):1101-1106.Zakova J et al. (2008)Human Reproduction 23, Suppl 1:P-333.Reproductive Low Improve ART Research into embryo Casals G et al. (2008)immunology outcomes. implantation and the Reproductive Biomedicine Onlinefunction of immune system 16(6):808–816.factors during pregnancy. Fukui A. et al. (2008)Fertility and Sterility 89(1):157-165.Grewal S et al. (2008)Proc Natl Acad Sci USA 105:16189-16194.Gremlich S. et al. (2008)Human Reproduction 23(5):1200-1206.Hapangama D et al. (2008)Human Reproduction 23,Suppl 1:O-207, i84.Hiby S. et al. (2008)Human Reproduction 23(4):972-976.Peng S. et al. (2008)Reproduction 135(3):367-375.Stoikos et al. (2008)Human Reproduction 23(6):1447-1456.Xu B et al. (2008)Human Reproduction 23(6):1394-1406.Yang H et al. (2008)Fertility and Sterility 89(3):656-661.Zhou W-H et al. (2008)Reproduction 135(3):385-395.Human Fertilisation & Embryology AuthorityScientific Horizon Scanning at the HFEA

26Section 3Issues identified in the 2008/9horizon scanning processIVF/ICSI technologies (continued)Issue Priority Use or aim Description Research identifiedControlled Low Refining Research into improving and Andersen et al. (2008)Ovarian the COH refining the COH protocol, Human Reproduction 23(6):1424-1430.Stimulation protocol and identifying markers of ovarian Binder H et al. (2008)(COH) prevention response and methods of Reproductive BioMedicine Onlineof ovarian predicting and reducing 17(2):185-189.hyper- the risk of OHSS. Busso C et al. (2008)stimulation Human Reproduction 23,syndrome Suppl 1:O-149, i60.(OHSS). Engmann L et al (2008)Fertility and Sterility 89(1):84-91.Fong S et al. (2008)Reproductive BioMedicine Online16(5):664-670.Gome-Palomares J et al (2008)Fertility and Sterility 89(3):620-624.Homburg R (2008)Human Fertility 11(1):17-22.Schoolcraft W et al (2008)Fertility and Sterility 89(1):151-156.Soares S et al. (2008)Human Reproduction Update14(4):321-333.Tsung-Hsien Lee et al. (2008)Human Reproduction 23(1):160-167.The Thessaloniki ESHRE/ ASRMsponsoredPCOS consensusworkshop group. (2008)Human Reproduction 23(3):462-477.Weghofer A. (2008)Human Reproduction 23(3):499-503.Miscarriage Low Reduce Identification of indicators of Cocksedge K. (2008)indicator miscarriage miscarriage – elevated free Human Reproduction 23(4):797-802.rate. androgen index and levels of Whitcomb B et al. (2008)cytokine thrombopoietin Fertility and Sterility 89(6):1795-1802.circulating in maternal blood.Sibling Low Improve Sibling embryos from the Yilmaz S et al. (2008)embryo ART same cycle have similar Reproductive Biomedicine Online 16(1):behaviour outcomes. developmental potential. 124-128.Development in vitro of ablastocyst can predictpregnancy outcome ofsibling embryo.Human Fertilisation & Embryology AuthorityScientific Horizon Scanning at the HFEA

Section 3Issues identified in the 2008/9horizon scanning process27IVF/ICSI technologies (continued)Issue Priority Use or aim Description Research identifiedICSI Low Improve ICSI Outline of risks and technical ESHRE Capri Workshop Group (2007)technique difficulties and indicators of Human Reproduction Updateand success. 113(6):515-526.outcomes. Nicopoullos J et al. (2008)Human Reproduction 23(2):240-250.Varghese A C et al. (2007)Reproductive Biomedicine Online15(6):719-727.Novel Low Improve ART Novel methods of embryo Goto S et al. (2007)transfer outcomes. transfer to increase Fertility and Sterility 88(5):1339-1343.methods pregnancy rate. Korosec S et al. (2007)Reproductive Biomedicine Online15(6):701-707.Markers of Low Predicting IVF High sIL2R and low LIF Gremlich S et al. (2008)IVF success success. maternal plasma levels on Human Reproduction 23,day of embryo transfer Suppl 1:P-345.associated with rapidpregnancy loss after IVF.Marker of Low Improvement Novel biomarker for male Steger K et al. (2008)male infertility of male infertility found in testicular Human Reproduction 23(1):11-16.infertility spermatids and ejaculateddiagnosis. spermatozoa.Chromosome Low Identify risks Indication of a higher than Feng C et al. (2008)mutation associated normal rate of de novo Fertility and Sterility 90(1):92-96.after ART with ART. mutations in the Ychromosome of childrencreated via ART.Human Fertilisation & Embryology AuthorityScientific Horizon Scanning at the HFEA

28Section 3Issues identified in the 2008/9horizon scanning processOtherIssue Priority Use or aim Description Research identifiedGene therapy High Treatment of Research into gene therapy Ghadami M et al. (2008)infertility and to treat infertility and stable Molecular Human Reproductionresearch into gene transfer in human 14(1):9-15.human embryonic stem cells. Shimizu T et al. (2007)embryonic Reproduction 134:677-682.stem cells Kojima Y et al. (2008)Fertility and Sterility 89,Suppl 3:1448-1454.Wilber A et al (2007)Stem Cell 25(11):2919-2927.Fetal cells in Medium Source of Fetal cells in the maternal Mikhail M et al. (2008)maternal primitive blood are primitive stem cells Human Reproduction 23(4):928-933.blood stem cells with the potential for widedifferentiation.Epigenetic in Medium Information Review of evidence suggests Feliciano M et al. (2008)ART and on safety of that ARTs and/or infertility Human Reproduction 23,infertility ART may affect epigenetic Suppl 1:O-27, i12.processes leading to Huntriss J et al. (2008)epigenetic diseases. Human Fertility 11(2):85-94.Marques C et al. (2008)Human Reproduction 23,Suppl 1:O-72, i30.Xeno-free Low Human Xeno-free cryopreservation Valbuena D et al. (2008).cryo- embryonic of human embryonic stem Reproductive BioMedicine Onlinepreservation stem cell cells, a slow freeze-rapid 17(1):127-135.of human storage thawing technique.embryonicstem cellsHuman Fertilisation & Embryology AuthorityScientific Horizon Scanning at the HFEA

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