. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Abstract citation ID: dead093.1049 P-730 Detection of mitochondrial reversal following meiotic spindle transfer: a finding of importance for mitochondrial replacement therapies used for the purpose of avoiding mtDNA disease transmission K. Spath 1 , N. Costa-Borges 2 , E. Nikitos 3 , K. Kostaras 3 , G.C. Caldero ´n 2 , P. Psathas 3 , D. Wells 4 1 Juno Genetics, Research Team, Oxford, United Kingdom 2 Embryotools, Embryology, Barcelona, Spain 3 The Institute of Life, IVF Clinic, Athens, Greece 4 Juno Genetics/University of Oxford, Nuffield Department of Women’s and Reproductive Health, Oxford, United Kingdom Study question: Following transfer of the meiotic spindle from a patient’s oocyte into an enucleated donor oocyte, do relative levels of patient and do- nor mtDNA remain stable? Summary answer: mtDNA transferred along with the patient’s spindle typi- cally remain at a low level. However, disproportionate expansion of the patient’s mtDNA can occur in some cases. What is known already: Mitochondrial DNA disorders are caused by mutations in the mitochondrial genome, disrupting ATP production. They have few treatment options and no cure. All mitochondria are derived from the oocyte, therefore mtDNA disorders are maternally inherited. It has been proposed that disease transmission could be avoided if female mtDNA muta- tion carriers underwent meiotic spindle transfer (MST), removing the chro- mosomes (on the spindle) from an affected oocyte and transferring them into the healthy cytoplasm of a donor oocyte. However, research using embryonic stem cells has suggested that the small population of mitochondria transferred along with the spindle can sometimes undergo dramatic expansion. Study design, size, duration: 25 infertile couples were enrolled in a pro- spective pilot study evaluating MST as a treatment for infertility. The female participants had a mean age of 37.2, and an average of 6.4 previous unsuc- cessful IVF cycles (range 3-11) characterised by extremely poor embryo de- velopment and without any previous pregnancy. Importantly, none of the patients were carriers of a mtDNA disorder. Oocyte donors with previous successful IVF outcomes were matched with patients according to standard practice. Participants/materials, setting, methods: Metaphase-II-spindles from pa- tient oocytes were transferred into enucleated donor oocytes. Reconstructed oocytes underwent ICSI and the resulting embryos were transferred at the blastocyst stage. Mitochondrial genomes were sequenced to identify polymor- phisms differing between the patient and oocyte donor. These variations were quantified in the embryos (blastocyst biopsies), during pregnancy (am- niocentesis), in newborns (cord blood, cord tissue, urine), at 3-6 months and at one year (saliva, urine, blood), revealing the relative amounts of each mito- chondrial type. Main results and the role of chance: This MST pilot study resulted in the birth of six children, indicating that the procedure is compatible with the pro- duction of viable embryos, capable of producing healthy live births. The patient’s mtDNA was shown to represent <1% of the total in all blastocysts produced, confirming that MST is highly reproducible and that relatively few mitochondria are transferred along with the spindle. For five of the six chil- dren, the proportion of the total mtDNA attributable to the patient appeared to be stable, remaining at very low levels in all of the samples from later de- velopmental stages. However, in one child the small quantity of mtDNA transferred along with the spindle increased disproportionately with respect to the mtDNA of the oocyte donor, ultimately representing 30-60% of the total at birth, depending on the tissue tested. The precise timing of the ex- pansion of one type of mtDNA at the expense of the other is not known but occurred sometime between the blastocyst stage and birth. By the time of birth, the levels of donor and patient mtDNA appeared to have stabilised (no further increases were seen at 6 months and one year). All of the children born remain developmentally normal and healthy. Limitations, reasons for caution: After using MST, several pregnancies were achieved for patients with a long history of unsuccessful IVF attempts, associated with poor oocyte quality and a failure to produce blastocysts. However, this small pilot study lacked the controls necessary for a definitive evaluation of MST as a tool for infertility treatment. Wider implications of the findings: All children born following MST were healthy. However, our results clearly demonstrate that a substantial degree of mtDNA ‘reversal’ is possible. Consequently, mitochondrial replacement ther- apies used for avoidance of mtDNA disorders might not always be successful, even when initial levels of mutant mtDNA in reconstructed oocytes are very low. Trial registration number: ISRCTN11455145 Abstract citation ID: dead093.1050 P-731 The accuracy of truly non-invasive PGT using spent culture media is insufficient to justify routine clinical use C. Avila Perez 1,2 , L. Parnell 3 , M. Florensa Bargallo 4 , J. Herreros Cuesta 4 , Z. Larreategui Laiseca 5 , N. Prados Dodd 6 , M. Ruiz Perez 7 , D. Wells 1,2 1 University of Oxford, Nuffield Department of Women’s and Reproductive Health, Oxford, United Kingdom 2 Juno Genetics, Clinical Genetics, Oxford, United Kingdom 3 University of Manchester, Maternal and Foetal Health Research Centre, Manchester, United Kingdom 4 IVI Barcelona, IVF Laboratory, Barcelona, Spain 5 IVI Bilbao, IVF Laboratory, Bilbao, Spain 6 IVI Seville, Medical Affairs, Seville, Spain 7 IVI Seville, IVF Laboratory, Seville, Spain Study question: Can non-invasive PGT (niPGT), using a protocol that avoids manipulation of the embryo before spent culture medium is collected, accu- rately determine embryo chromosomal status? Summary answer: Accuracy using a truly non-invasive PGT method was low, suggesting this strategy is not appropriate for clinical use and should be restricted to research studies. What is known already: PGT requires embryo biopsy to obtain small num- bers of cells for genetic testing. The procedure requires skilled personnel and specialist equipment, significantly adding to the cost of PGT while also creat- ing training and logistical bottlenecks that reduce patient access to PGT. Additionally, there have been concerns that biopsy could damage embryos. These considerations have stimulated interest in potential non-invasive PGT strategies. Thus far, niPGT methods have mostly focused on cell-free embry- onic DNA found in spent culture media, but studies reporting highest accu- racy have tended to involve embryo manipulations likely to promote DNA re- lease and therefore cannot be considered truly non-invasive. Study design, size, duration: Samples of spent culture medium (SCM) as- sociated with 128 embryos were collected. Oocytes had been denuded of cu- mulus cells, fertilised using ICSI and the resulting embryos thoroughly washed on day-4 before transfer to a fresh drop of medium. SCM samples were col- lected on day-5 or day-6 of culture. Embryos had not previously been cryo- preserved and underwent minimal manipulation prior to SCM collection. Results of SCM analysis were compared to those obtained following conven- tional ‘invasive’ PGT. Participants/materials, setting, methods: Media samples were subjected to whole genome amplification using the PG-Seq Rapid Non-Invasive PGT kit. Concentrations of sequencing libraries were measured, and next-generation sequencing (NGS) was undertaken. The resulting NGS data was analysed with PG-Find Software to predict chromosomal status. In parallel, trophecto- derm biopsies from the corresponding embryos were analysed using a well- established and highly validated PGT method, based upon amplification of thousands of sites across the genome, followed by NGS. Main results and the role of chance: Despite the implementation of rigor- ous measures to prevent contamination of SCM samples, extraneous female DNA, likely of cumulus cell origin, was frequently observed. Depending on the clinic, 16.7% to 38.1% of media samples associated with male embryos gave a discordant (female) result. This highlights the difficulty of avoiding inad- vertent sampling of maternal DNA. There were no instances of male DNA contamination affecting SCM samples from female embryos. We assessed 39 th Hybrid Annual Meeting of the ESHRE, Copenhagen – Denmark, 25-28 June 2023 i533 Downloaded from https://academic.oup.com/humrep/article/38/Supplement_1/dead093.1049/7202846 by guest on 24 June 2023