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STUDY QUESTION How should ART/preimplantation genetic testing (PGT) centres manage the detection of chromosomal mosaicism following PGT? SUMMARY ANSWER Thirty good practice recommendations were formulated that can be used by ART/PGT centres as a basis for their own policy with regards to the management of ‘mosaic’ embryos. WHAT IS KNOWN ALREADY The use of comprehensive chromosome screening technologies has provided a variety of data on the incidence of chromosomal mosaicism at the preimplantation stage of development and evidence is accumulating that clarifies the clinical outcomes after transfer of embryos with putative mosaic results, with regards to implantation, miscarriage and live birth rates, and neonatal outcomes. STUDY DESIGN, SIZE, DURATION This document was developed according to a predefined methodology for ESHRE good practice recommendations. Recommendations are supported by data from the literature, a large survey evaluating current practice and published guidance documents. The literature search was performed using PubMed and focused on studies published between 2010 and 2022. The survey was performed through a web-based questionnaire distributed to members of the ESHRE special interest groups (SIG) Reproductive Genetics and Embryology, and the ESHRE PGT Consortium members. It included questions on ART and PGT, reporting, embryo transfer policy and follow-up of transfers. The final dataset represents 239 centres. PARTICIPANTS/MATERIALS, SETTING, METHODS The working group (WG) included 16 members with expertise on the ART/PGT process and chromosomal mosaicism. The recommendations for clinical practice were formulated based on the expert opinion of the WG, while taking into consideration the published data and results of the survey. MAIN RESULTS AND THE ROLE OF CHANCE Eighty percent of centres that biopsy three or more cells report mosaicism, even though only 66.9% of all centres have validated their technology and only 61.8% of these have validated specifically for the calling of chromosomal mosaicism. The criteria for designating mosaicism, reporting and transfer policies vary significantly across the centres replying to the survey. The WG formulated recommendations on how to manage the detection of chromosomal mosaicism in clinical practice, considering validation, risk assessment, designating and reporting mosaicism, embryo transfer policies, prenatal testing and follow-up. Guidance is also provided on the essential elements that should constitute the consent forms and the genetic report, and that should be covered in genetic counselling. As there are several unknowns in chromosomal mosaicism, it is recommended that PGT centres monitor emerging data on the topic and adapt or refine their policy whenever new insights are available from evidence. LIMITATIONS, REASONS FOR CAUTION Rather than providing instant standardized advice, the recommendations should help ART/PGT centres in developing their own policy towards the management of putative mosaic embryos in clinical practice. WIDER IMPLICATIONS OF THE FINDINGS This document will help facilitate a more knowledge-based approach for dealing with chromosomal mosaicism in different centres. In addition to recommendations for clinical practice, recommendations for future research were formulated. Following up on these will direct research towards existing research gaps with direct translation to clinical practice. Emerging data will help in improving guidance, and a more evidence-based approach of managing chromosomal mosaicism. STUDY FUNDING/COMPETING INTEREST(S) The WG received technical support from ESHRE. M.D.R. participated in the EQA special advisory group, outside the submitted work, and is the chair of the PGT WG of the Belgian society for human genetics. D.W. declared receiving salary from Juno Genetics, UK. A.C. is an employee of Igenomix, Italy and C.R. is an employee of Igenomix, Spain. C.S. received a research grant from FWO, Belgium, not related to the submitted work. I.S. declared being a Co-founder of IVFvision Ltd, UK. J.R.V. declared patents related to ‘Methods for haplotyping single-cells’ and ‘Haplotyping and copy number typing using polymorphic variant allelic frequencies’, and being a board member of Preimplantation Genetic Diagnosis International Society (PGDIS) and International Society for Prenatal Diagnosis (ISPD). K.S. reported being Chair-elect of ESHRE. The other authors had nothing to disclose. DISCLAIMER This Good Practice Recommendations (GPR) document represents the views of ESHRE, which are the result of consensus between the relevant ESHRE stakeholders and are based on the scientific evidence available at the time of preparation.  ESHRE GPRs should be used for information and educational purposes. They should not be interpreted as setting a standard of care or be deemed inclusive of all proper methods of care, or be exclusive of other methods of care reasonably directed to obtaining the same results. They do not replace the need for application of clinical judgement to each individual presentation, or variations based on locality and facility type.  Furthermore, ESHRE GPRs do not constitute or imply the endorsement, or favouring, of any of the included technologies by ESHRE. 

The field of preimplantation genetic testing (PGT) is evolving fast, and best practice advice is essential for regulation and standardisation of diagnostic testing. The previous ESHRE guidelines on best practice for PGD, published in 2005 and 2011, are considered outdated, and the development of new papers outlining recommendations for good practice in PGT was necessary. The current paper provides recommendations on the technical aspects of PGT for chromosomal structural rearrangements (PGT-SR) and PGT for aneuploidies (PGT-A) and covers recommendations on array-based comparative genomic hybridisation (aCGH) and next-generation sequencing (NGS) for PGT-SR and PGT-A and on fluorescence in situ hybridisation (FISH) and single nucleotide polymorphism (SNP) array for PGT-SR, including laboratory issues, work practice controls, pre-examination validation, preclinical work-up, risk assessment and limitations. Furthermore, some general recommendations on PGT-SR/PGT-A are formulated around training and general risk assessment, and the examination and post-examination process. This paper is one of a series of four papers on good practice recommendations on PGT. The other papers cover the organisation of a PGT centre, embryo biopsy and tubing and the technical aspects of PGT for monogenic/single-gene defects (PGT-M).Together, these papers should assist everyone interested in PGT in developing the best laboratory and clinical practice possible.

Background Mitochondrial or oxidative phosphorylation diseases are relatively frequent, multisystem disorders; in about 15% of cases they are caused by maternally inherited mitochondrial DNA (mtDNA) mutations. Because of the possible severity of the phenotype, the lack of effective treatment, and the high recurrence risk for offspring of carrier females, couples wish to prevent the transmission of these mtDNA disorders to their offspring. Prenatal diagnosis is problematic for several reasons, and concern the often poor correlation between mutation percentages and disease severity and the uncertainties about the representativeness of a fetal sample. A new option for preventing transmission of mtDNA disorders is preimplantation genetic diagnosis (PGD), which circumvents these problems by transferring an embryo below the threshold of clinical expression. Methods We present the data on nine PGD cycles in four female carriers of mitochondrial diseases: three mitochondrial encephalopathy, lactic acidosis and stroke-like episodes (MELAS) (m.3243A>G), and one Leigh (m.8993T>G). Our threshold for transfer after PGD is 15% for the m.3243A>G mutation and 30% for the m.8993T>G mutation. Results All four female carriers produced embryos eligible for transfer. The m.8993T>G mutation in oocytes/embryos showed more skewing than the m.3243A>G. In about 80% of the embryos the mutation load in the individual blastomeres was fairly constant (interblastomere differences <10%). However, in around 11% (in embryos with the m.3243A>G mutation only), the mutation load differed substantially (>15%) between blastomeres of a single embryo, mostly as a result of one outlier. The m.8993T>G carrier became pregnant and gave birth to a healthy son. Conclusions PGD provides carriers of mtDNA mutations the opportunity to conceive healthy offspring.

The field of preimplantation genetic testing (PGT) is evolving fast, and best practice advice is essential for regulation and standardisation of diagnostic testing. The previous ESHRE guidelines on best practice for preimplantation genetic diagnosis, published in 2005 and 2011, are considered outdated and the development of new papers outlining recommendations for good practice in PGT was necessary.The current updated version of the recommendations for good practice is, similar to the 2011 version, split into four documents, one of which covers the organisation of a PGT centre. The other documents focus on the different technical aspects of embryo biopsy, PGT for monogenic/single-gene defects (PGT-M) and PGT for chromosomal structural rearrangements/aneuploidies (PGT-SR/PGT-A).The current document outlines the steps prior to starting a PGT cycle, with details on patient inclusion and exclusion, and counselling and information provision. Also, recommendations are provided on the follow-up of PGT pregnancies and babies. Finally, some further recommendations are made on the practical organisation of an IVF/PGT centre, including basic requirements, transport PGT and quality management.This document, together with the documents on embryo biopsy, PGT-M and PGT-SR/PGT-A, should assist everyone interested in PGT in developing the best laboratory and clinical practice possible.

Purpose: Our aim was to compare the accuracy of family- or disease-specific targeted haplotyping and direct mutation-detection strategies with the accuracy of genome-wide mapping of the parental origin of each chromosome, or karyomapping, by single-nucleotide polymorphism genotyping of the parents, a close relative of known disease status, and the embryo cell(s) used for preimplantation genetic diagnosis of single-gene defects in a single cell or small numbers of cells biopsied from human embryos following in vitro fertilization. Methods: Genomic DNA and whole-genome amplification products from embryo samples, which were previously diagnosed by targeted haplotyping, were genotyped for single-nucleotide polymorphisms genome-wide detection and retrospectively analyzed blind by karyomapping. Results: Single-nucleotide polymorphism genotyping and karyomapping were successful in 213/218 (97.7%) samples from 44 preimplantation genetic diagnosis cycles for 25 single-gene defects with various modes of inheritance distributed widely across the genome. Karyomapping was concordant with targeted haplotyping in 208 (97.7%) samples, and the five nonconcordant samples were all in consanguineous regions with limited or inconsistent haplotyping results. Conclusion: Genome-wide karyomapping is highly accurate and facilitates analysis of the inheritance of almost any single-gene defect, or any combination of loci, at the single-cell level, greatly expanding the range of conditions for which preimplantation genetic diagnosis can be offered clinically without the need for customized test development. Genet Med 16 11, 838–845.

High-throughput sequencing technologies have increasingly led to discovery of disease-causing genetic variants, primarily in postnatal multi-cell DNA samples. However, applying these technologies to preimplantation genetic testing (PGT) in nuclear or mitochondrial DNA from single or few-cells biopsied from in vitro fertilised (IVF) embryos is challenging. PGT aims to select IVF embryos without genetic abnormalities. Although genotyping-by-sequencing (GBS)-based haplotyping methods enabled PGT for monogenic disorders (PGT-M), structural rearrangements (PGT-SR), and aneuploidies (PGT-A), they are labour intensive, only partially cover the genome and are troublesome for difficult loci and consanguineous couples. Here, we devise a simple, scalable and universal whole genome sequencing haplarithmisis-based approach enabling all forms of PGT in a single assay. In a comparison to state-of-the-art GBS-based PGT for nuclear DNA, shallow sequencing-based PGT, and PCR-based PGT for mitochondrial DNA, our approach alleviates technical limitations by decreasing whole genome amplification artifacts by 68.4%, increasing breadth of coverage by at least 4-fold, and reducing wet-lab turn-around-time by ~2.5-fold. Importantly, this method enables trio-based PGT-A for aneuploidy origin, an approach we coin PGT-AO, detects translocation breakpoints, and nuclear and mitochondrial single nucleotide variants and indels in base-resolution. This work demonstrates a novel preimplantation genetic testing (PGT) method that detects aberrations in both the nuclear and mitochondrial genomes of IVF embryos. The technology traces the origin of chromosomal aberrations to before or after fertilisation.

Neurofibromatosis type 1 (NF1) is an autosomal dominant disorder that affects the skin and the nervous system. The condition is completely penetrant with extreme clinical variability, resulting in unpredictable manifestations in affected offspring, complicating reproductive decision-making. One of the reproductive options to prevent the birth of affected offspring is preimplantation genetic testing (PGT). We performed a retrospective review of the medical files of all couples (n = 140) referred to the Dutch PGT expert center with the indication NF1 between January 1997 and January 2020. Of the couples considering PGT, 43 opted out and 15 were not eligible because of failure to identify the underlying genetic defect or unmet criteria for in vitro fertilization (IVF) treatment. The remaining 82 couples proceeded with PGT. Fertility assessment prior to IVF treatment showed a higher percentage of male infertility in males affected with NF1 compared to the partners of affected females. Cardiac evaluations in women with NF1 showed no contraindications for IVF treatment or pregnancy. For 67 couples, 143 PGT cycles were performed. Complications of IVF treatment were not more prevalent in affected females compared to partners of affected males. The transfer of 174 (out of 295) unaffected embryos led to 42 ongoing pregnancies with a pregnancy rate of 24.1% per embryo transfer. There are no documented cases of misdiagnosis following PGT in this cohort. With these results, we aim to provide an overview of PGT for NF1 with regard to success rate and safety, to optimize reproductive counseling and PGT treatment for NF1 patients.

PurposeWe aim to evaluate the safety of PGD. We focus on the congenital malformation rate and additionally report on adverse perinatal outcome.MethodsWe collated data from a large group of singletons and multiples born after PGD between 1995 and 2014. Data on congenital malformation rates in live born children and terminated pregnancies, misdiagnosis rate, birth parameters, perinatal mortality, and hospital admissions were prospectively collected by questionnaires.ResultsFour hundred thirty-nine pregnancies in 381 women resulted in 364 live born children. Nine children (2.5%) had major malformations. This percentage is consistent with other PGD cohorts and comparable to the prevalence reported by the European Surveillance of Congenital Anomalies (EUROCAT). We reported one misdiagnosis resulting in a spontaneous abortion of a fetus with an unbalanced chromosome pattern. 20% of the children were born premature (< 37 weeks) and less than 15% had a low birth weight. The incidence of hospital admissions is in line with prematurity and low birth weight rate. One child from a twin, one child from a triplet, and one singleton died at 23, 32, and 37 weeks of gestation respectively.ConclusionsWe found no evidence that PGD treatment increases the risk on congenital malformations or adverse perinatal outcome.Trial registration numberNCT 2 149485

BackgroundPreimplantation genetic diagnosis (PGD) is a reproductive strategy for mitochondrial DNA (mtDNA) mutation carriers, strongly reducing their risk of affected offspring. Embryos either without the mutation or with mutation load below the phenotypic threshold are transferred to the uterus. Because of incidental heteroplasmy deviations in single blastomere and the relatively limited data available, we so far preferred relying on two blastomeres rather than one. Considering the negative effect of a two-blastomere biopsy protocol compared with a single-blastomere biopsy protocol on live birth delivery rate, we re-evaluated the error rate in our current dataset.MethodsFor the m.3243A>G mutation, sufficient embryos/blastomeres were available for a powerful analysis. The diagnostic error rate, defined as a potential false-negative result, based on a threshold of 15%, was determined in 294 single blastomeres analysed in 73 embryos of 9 female m.3243A>G mutation carriers.ResultsOnly one out of 294 single blastomeres (0.34%) would have resulted in a false-negative diagnosis. False-positive diagnoses were not detected.ConclusionOur findings support a single-blastomere biopsy PGD protocol for the m.3243A>G mutation as the diagnostic error rate is very low. As in the early preimplantation embryo no mtDNA replication seems to occur and the mtDNA is divided randomly among the daughter cells, we conclude this result to be independent of the specific mutation and therefore applicable to all mtDNA mutations.

Preimplantation genetic diagnosis (PGD) for chromosomal rearrangements (CR) is mainly based on fluorescence in situ hybridisation (FISH). Application of this technique is limited by the number of available fluorochromes, the extensive preclinical work-up and technical and interpretative artefacts. We aimed to develop a universal, off-the-shelf protocol for PGD by combining single-nucleotide polymorphism (SNP) array-derived copy number (CN) determination and genotyping for detection of unbalanced translocations in cleavage-stage embryos. A total of 36 cleavage-stage embryos that were diagnosed as unbalanced by initial PGD FISH analysis were dissociated (n=146) and amplified by multiple displacement amplification (MDA). SNP CNs and genotypes were determined using SNP array. Epstein-Barr Virus-transformed cell lines with known CR were used for optimising the genomic smoothing (GS) length setting to increase signal to noise ratio. SNP CN analysis showed 23 embryos (64%) that were unbalanced in all blastomeres for the chromosomes involved in the translocation, 5 embryos (14%) that were normal or balanced in all blastomeres and 8 embryos (22%) that were mosaic. SNP genotyping, based on analysis of informative SNP loci with opposing homozygous parental genotypes, confirmed partial monosomies associated with inheritance of unbalanced translocation in surplus embryos. We have developed a universal MDA-SNP array technique for chromosome CN analysis in single blastomeres. SNP genotyping could confirm partial monosomies. This combination of techniques showed improved diagnostic specificity compared with FISH and may provide more reliable PGD analysis associated with higher embryo transfer rate.