O-051 Ethical and counseling considerations

To evaluate whether increasing total gonadotropin (Gn) dose is associated with changes in euploid blastocyst rate in preimplantation genetic testing (PGT) oocytes. This retrospective cohort study was conducted between 2017 and 2022, and 19,246 oocytes were grouped and analyzed based on tri-sectional quantiles of total Gn doses. Single reproductive medical center. All the patients who underwent PGT cycles, including PGT for aneuploidy, monogenic disorders, and structural rearrangements, were included. Next-generation sequencing platforms for chromosomal analysis. Blastocyst formation and euploid blastocyst rates. In total, 19,246 oocytes and 5375 PGT blastocysts were analyzed. There were significant differences in blastocyst formation and euploid blastocyst rates among the groups classified according to tri-sectional quantiles of total Gn doses. Significant differences in age, body mass index (BMI), proportion of primary infertility, anti-Müllerian hormone (AMH) levels, number of oocytes retrieved, controlled ovarian stimulation (COS) regimen, type of Gn, and PGT category were observed among the three groups. After stratifying the analysis by age, BMI, infertility diagnosis, AMH levels, number of oocytes retrieved, PGT category, type of Gn, and COS regimen, significant differences were only seen in a small number of specific subgroups. Furthermore, the results of the multiple logistic regression analysis showed that the blastocyst formation and euploid blastocyst rates did not significantly increase or decrease with the total Gn dose, whether treated as a continuous variable or divided into three Gn groups as categorical variables. Notably, advancing age was a risk factor for blastocyst formation and euploid blastocyst rates. PGT for structural rearrangements was a risk factor for blastocyst formation and euploid blastocyst rates as compared with PGT for aneuploidy. In the total PGT cycles, advancing age, and preimplantation genetic testing for structural rearrangements negatively affected blastocyst formation and euploid blastocyst rates; however, the total Gn dose did not affect blastocyst formation and euploid blastocyst rates.

What are the trends and developments in preimplantation genetic testing (PGT) in 2018 as compared to previous years? The main trends observed in this 21st dataset on PGT are that the implementation of trophectoderm biopsy with comprehensive whole-genome testing is most often applied for PGT-A and concurrent PGT-M/SR/A, while for PGT-M and PGT-SR, single-cell testing with PCR and FISH still prevail. Since it was established in 1997, the ESHRE PGT Consortium has been collecting and analysing data from mainly European PGT centres. To date, 20 datasets and an overview of the first 10 years of data collections have been published. The data for PGT analyses performed between 1 January 2018 and 31 December 2018 with a 2-year follow-up after analysis were provided by participating centres on a voluntary basis. Data were collected using an online platform, which is based on genetic analysis and has been in use since 2016. Data on biopsy method, diagnostic technology, and clinical outcome were submitted by 44 centres. Records with analyses for more than one PGT for monogenic disorders (PGT-M) and/or PGT for chromosomal structural rearrangements (PGT-SR), or with inconsistent data regarding the PGT modality, were excluded. All transfers performed within 2 years after the analysis were included, enabling the calculation of cumulative pregnancy rates. Data analysis, calculations, and preparation of figures and tables were carried out by expert co-authors. The current data collection from 2018 covers a total of 1388 analyses for PGT-M, 462 analyses for PGT-SR, 3003 analyses for PGT for aneuploidies (PGT-A), and 338 analyses for concurrent PGT-M/SR with PGT-A.The application of blastocyst biopsy is gradually rising for PGT-M (from 19% in 2016-2017 to 33% in 2018), is status quo for PGT-SR (from 30% in 2016-2017 to 33% in 2018) and has become the most used biopsy stage for PGT-A (from 87% in 2016-2017 to 98% in 2018) and for concurrent PGT-M/SR with PGT-A (96%). The use of comprehensive, whole-genome amplification (WGA)-based diagnostic technology showed a small decrease for PGT-M (from 15% in 2016-2017 to 12% in 2018) and for PGT-SR (from 50% in 2016-2017 to 44% in 2018). Comprehensive testing was, however, the main technology for PGT-A (from 93% in 2016-2017 to 98% in 2018). WGA-based testing was also widely used for concurrent PGT-M/SR with PGT-A, as a standalone technique (74%) or in combination with PCR or FISH (24%). Trophectoderm biopsy and comprehensive testing strategies are linked with higher diagnostic efficiencies and improved clinical outcomes per embryo transfer. The findings apply to the data submitted by 44 participating centres and do not represent worldwide trends in PGT. Details on the health of babies born were not provided in this manuscript. The Consortium datasets provide a valuable resource for following trends in PGT practice. The study has no external funding, and all costs are covered by ESHRE. There are no competing interests declared. N/A.

Initially categorized 46,XY embryo transfer ending with 45,X products of conception—a case report and a review of discordant result management

Background: Neurofibromatosis is an autosomal dominant genetic disease caused by the abnormal development of neural crests due to genetic defects and is difficult to treat. Patients have a characteristic phenotype with neurofibromas as the main features in different forms, which are accompanied by multisystem involvement. The clinical symptoms of this disease vary greatly, making the treatment more difficult. Methods: Preimplantation genetic testing (PGT) is a useful technique to prevent chromosomal aneuploidies and other genetic disorders in origin. PGT for monogenic diseases (PGT-M) is now widely used as an effective strategy to screen embryos for monogenic or chromosomal diseases before implantation. In this study, PGT-M was performed in a family history of hereditary with neurofibromatosis type 1 (NF1) to prevent the offspring from inheriting disease-causing gene variant from their parents. Trio-based whole-exome sequencing was used to identify potential pathogenic variants associated with NF1. Blastocyst biopsy was performed on embryos obtained by intracytoplasmic sperm injection. Single-cell amplification of biopsied cells was performed for targeted next-generation sequencing. Single nucleotide polymorphism markers on both sides of NF1 were selected to identify disease-carrying haplotypes in each embryo. Results: A novel heterozygotic frameshift pathogenic variant, c.2033_2034delinsA(p.P678Qfs*10), was identified in the NF1 gene in the proband. A total of five blastocysts were biopsied, and the PGT results showed that only one blastocyst was unaffected and was euploid, and the remaining four blastocysts were all carrying paternal pathogenic variants. The only one normal blastocyst was transferred in a frozen-thawed embryo transfer cycle, and a live singleton pregnancy was successfully achieved. At 18 weeks, the amniocentesis test revealed normal karyotype, and the variant carried by the proband was not detected. At 40 weeks, the proband's wife successfully delivered a healthy baby naturally. Conclusion: PGT is an effective method to detect chromosome copy number variation and gene variant sites in embryos, and it provides suggestions for possible innovations to block the transmission of single-gene genetic diseases to offspring, thereby preventing the occurrence of birth defects.

Can whole genome sequencing (WGS) offer a relatively cost-effective approach for embryonic genome-wide haplotyping and preimplantation genetic testing (PGT) for monogenic disorders (PGT-M), aneuploidy (PGT-A) and structural rearrangements (PGT-SR)? Reliable genome-wide haplotyping, PGT-M, PGT-A and PGT-SR could be performed by WGS with 10× depth of parental and 4× depth of embryonic sequencing data. Reduced representation genome sequencing with a genome-wide next-generation sequencing haplarithmisis-based solution has been verified as a generic approach for automated haplotyping and comprehensive PGT. Several low-depth massively parallel sequencing (MPS)-based methods for haplotyping and comprehensive PGT have been developed. However, an additional family member, such as a sibling, or a proband, is required for PGT-M haplotyping using low-depth MPS methods. In this study, 10 families that had undergone traditional IVF-PGT and 53 embryos, including 13 embryos from two PGT-SR families and 40 embryos from eight PGT-M families, were included to evaluate a WGS-based method. There were 24 blastomeres and 29 blastocysts in total. All embryos were used for PGT-A. Karyomapping validated the WGS results. Clinical outcomes of the 10 families were evaluated. A blastomere or a few trophectoderm cells from the blastocyst were biopsied, and multiple displacement amplification (MDA) was performed. MDA DNA and bulk DNA of family members were used for library construction. Libraries were sequenced, and data analysis, including haplotype inheritance deduction for PGT-M and PGT-SR and read-count analysis for PGT-A, was performed using an in-house pipeline. Haplotyping with a proband and parent-only haplotyping without additional family members were performed to assess the WGS methodology. Concordance analysis between the WGS results and traditional PGT methods was performed. For the 40 PGT-M and 53 PGT-A embryos, 100% concordance between the WGS and single-nucleotide polymorphism (SNP)-array results was observed, regardless of whether additional family members or a proband was included for PGT-M haplotyping. For the 13 embryos from the two PGT-SR families, the embryonic balanced translocation was detected and 100% concordance between WGS and MicroSeq with PCR-seq was demonstrated. The number of samples in this study was limited. In some cases, the reference embryo for PGT-M or PGT-SR parent-only haplotyping was not available owing to failed direct genotyping. WGS-based PGT-A, PGT-M and PGT-SR offered a comprehensive PGT approach for haplotyping without the requirement for additional family members. It provided an improved complementary method to PGT methodologies, such as low-depth MPS- and SNP array-based methods. This research was supported by the research grant from the National Key R&D Program of China (2018YFC0910201 and 2018YFC1004900), the Guangdong province science and technology project of China (2019B020226001), the Shenzhen Birth Defect Screening Project Lab (JZF No. [2016] 750) and the Shenzhen Municipal Government of China (JCYJ20170412152854656). This work was also supported by the National Natural Science Foundation of China (81771638, 81901495 and 81971344), the National Key R&D Program of China (2018YFC1004901 and 2016YFC0905103), the Shanghai Sailing Program (18YF1424800), the Shanghai Municipal Commission of Science and Technology Program (15411964000) and the Shanghai 'Rising Stars of Medical Talent' Youth Development Program Clinical Laboratory Practitioners Program (201972). The authors declare no competing interests. N/A.

Preimplantation genetic testing for structural rearrangement based on low-coverage next-generation sequencing accurately discriminates between normal and carrier embryos for patients with translocations

This study aimed to evaluate the rates of euploidy, aneuploidy, and mosaicism in preimplantation genetic testing for structural rearrangements (PGT-SR) cycles from chromosomal inversion carriers. In addition, this work also focused on assessing the impact of some contributors on the incidence of parental originating aneuploidy and mosaicism. This retrospective review enrolled chromosomal inversion carrier couples of whom the females were under 38years old undergoing PGT-SR at a single academic reproductive center. Subgroups were divided according to the gender of carriers, the inversion type, and the semen parameters of male carriers (male factor infertility (MF) or non-MF). Patient demographics, cycle characteristics, and PGT-SR outcomes were compared among subgroups. A total of 71 PGT-SR cycles from 57 inversion carrier couples were included for analysis. Among the 283 blastocysts, 48.4% were identified as euploidy, 27.9% as aneuploidy, and the remaining 23.7% as mosaicism. Only 32.9% of aneuploid embryos and 1.5% of mosaic embryos involved the parental inversion chromosomes. Notably, the female inversion carriers seemed to produce more parental originating aneuploid embryos than male inversion carriers (45.5% vs 23.9%, p = 0.044). The type of inversion and sperm parameters of male chromosomal inversion carriers did not affect the ploidy status of embryos. The incidence of parental originating aneuploidy in inversion carrier couples is lower than expected. For male chromosomal inversion carriers with normal sperm condition whose female partners are under 38years old, natural conception combined with prenatal diagnosis could be provided as an option during fertility counseling.

Study question How do genetic centres in Belgium apply international guidelines on preimplantation genetic testing (PGT)? Summary answer We present the national consensus on how to practice PGT, starting from initial patient uptake and embryo selection to follow-up of pregnancies. What is known already PGT includes PGT-M for monogenic disorders, PGT-SR for structural rearrangements and PGT-A for aneuploidy screening. The application of whole-genome technologies to trophectoderm biopsies for PGT-M and PGT-SR/A has evidenced the high prevalence of mosaicism due to chromosome segregation errors during the first mitotic divisions, making biopsy results interpretation and embryo transfer policy more challenging and complex. Guidelines from several international societies have been published in recent years to leverage the standardization of the whole PGT service. Although these are based on expertise and continuously acquiring evidence, it is widely recognized that variations in local and national regulations diversify PGT practices. Study design, size, duration Recommendations regarding PGT indications, patient counselling, embryo transfer policy, reporting, quality and pregnancy follow-up have been built to harmonize PGT services nationally. With a special focus on mosaicism, aiming to balance the highest chance for a pregnancy with the lowest risk for a chromosomally abnormal ongoing pregnancy or child. Participants/materials, setting, methods The Belgian PGT working group consists of laboratory geneticists, clinical geneticists and IVF specialists from eight nationally accredited centres. The group operates under the BeSHG umbrella and meets quarterly to formulate opinions and statements on PGT practice with the main goal of guaranteeing a high standard of care, exchange knowledge and implement a harmonized workflow. The Belgian social security system provides reimbursement of six genetic testing cycles for all PGT-M/SR patients but not for PGT-A. Main results and the role of chance PGT is offered for all monogenic and chromosomal disorders, if the causal genetic variants are known and documented. PGT for HLA-typing is allowed for the therapeutic benefit of an existing child after couple's psychological evaluation. Aneuploidy screening can be offered to specific patient groups to increase IVF success rate. However, social sexing and testing for non-medical or eugenic purposes is prohibited. At intake, the couple is counselled about the genetic aspect of the disease, psychological and financial aspects of PGT, applied methodology, including average success and misdiagnosis rate, and embryo transfer policies. Affected embryos, and embryos without diagnosis for PGT-M/SR are not transferred. For PGT-A (combined or not with PGT-M/SR), embryos are categorized as: (A) Euploid - suitable for transfer, if mosaicism is absent or below the detection threshold. (B) Mosaic transferable - suitable for transfer, but only following ranking and/or counselling and follow up, if mosaicism for any chromosome not described in (C) is detected; (C) Aneuploid or Mosaic - not suitable for transfer, if mosaicism of 8, 9, 13, 16, 18, 21 and X or full aneuploidy of any chromosome is detected. Non-invasive prenatal testing is strongly recommended to all women pregnant after PGT and is nationally reimbursed. Limitations, reasons for caution The practical recommendations, which are supplementary to the recently published ESHRE PGT recommendations, are based on expertise and data from the literature available at the time of preparation. As such, they will need regular reconsideration and an update or extension may follow when new solid scientific evidence arises. Wider implications of the findings As international recommendations represent a greatest common denominator over many local regulations, this work serves as example for drawing country-specific agreements for genetic centres. The process of consensus expert opinion formation on best practice may be applicable in other countries where PGT service is not overseen by a designated agency. Trial registration number Not applicable

Whole-genome analysis of a putative rare and complex interchromosomal reciprocal insertion: thorough investigations for a straightforward interpretation

Blastocyst conversion rate and ploidy in translocation carriers

Next-Generation Sequencing Is More Efficient at Detecting Mosaic Embryos and Improving Pregnancy Outcomes than Single-Nucleotide Polymorphism Array Analysis

Genomics Quality Assessment has provided external quality assessments (EQAs) for preimplantation genetic testing (PGT) for 12years for eight monogenic diseases to identify sub-optimal PGT strategies, testing and reporting of results, which can be shared with the genomics community to aid optimised standards of PGT services for couples. The EQAs were provided in two stages to mimic end-to-end protocols. Stage 1 involved DNA feasibility testing of a couple undergoing PGT and affected proband. Participants were required to report genotyping results and outline their embryo testing strategy. Lymphoblasts were distributed for mock embryo testing for stage 2. Submitted clinical reports and haplotyping results were assessed against peer-ratified criteria. Performance was monitored to identify poor performance. The most common testing methodology was short tandem repeat linkage analysis (59%); however, the adoption of single nucleotide polymorphism-based platforms was observed and a move from blastomere to trophectoderm testing. There was a variation in testing strategies, assigning marker informativity and understanding test limitations, some clinically unsafe. Critical errors were reported for genotyping and interpretation. EQA provides an overview of the standard of preimplantation genetic testing-M clinical testing and identifies areas of improvement for accurate detection of high-risk embryos.

Preimplantation genetic testing (PGT) is an alternative reproductive technology integrated with in vitro fertilization (IVF). It is a well-established technique offering a reproductive option for families at a high risk of transmitting a genetic disorder, allowing them to avoid making a decision about termination of an affected pregnancy (TOP). In Arab communities and particularly in Oman, where TOP is not favored under the majority of implemented Muslim law, termination of pregnancy for fetal indications is not always possible. As these communities are in favor of consanguineous marriage, they are at increased risk of serious and lethal autosomal recessive conditions, and as a result, PGT is a feasible option as a TOP decision can be avoided. However, undergoing PGT is relatively new in the Arab Muslim countries and Omani patients have only recently had access to the service. This qualitative study utilized a phenomenological approach to explore the experience of Omani families who had selected to undergo PGT as a means of reducing the risk of having a child affected with a genetic disorder. Fourteen participants from eight families who underwent PGT were interviewed. Data collected were analyzed using thematic analysis. The research identified four main themes: Anxiously waiting 'Taraqub'; Unforeseen; Secrecy; and Me and My Partner. The findings of the research have provided insight into the PGT experiences of Omani families. Unique cultural and religious perspectives should be considered when counseling Omani Muslim couples.

Introduction Pre- implantation Genetic Testing (PGT), including Pre- Implantation Genetic Screening (PGS) and Pre-Implantation Genetic Diagnosis (PGD) is defined as a group of genetic analysis applied to embryos produced through IVF programs. In Bahrain, these investigations were introduced since 2007, by referring embryo biopsy samples to PGD labs outside the country. Most of the tests were initially implemented using the techniques of FISH and PCR, while a-CGH and NGS emerged later. Genetic testing of embryos is laborious, costly, stressful and psychologically harsh on families. It should be organized and protected to ensure safety of testing and authenticity of outcome. Currently, the PGT practices in Bahrain and some of GCC countries lack many elements of quality management and require strategies for transformation of commercially oriented business to a patient-welfare oriented service. This study will present PGT data collected in the last 10 years from three major IVF centres in Bahrain. It will basically emphasize on the problems and the weaknesses encountered in our system through analysis of the data and treatment outcome in terms of pregnancy success rates. Also, in an endeavour to improve the quality of PGT services and to overcome some of the implementation-related issues observed, bioethical standards helping in improvement of the system and regulation of PGT practices will be proposed and discussed. Materials and Methods This study will present the findings of 500 infertility/ PGT cases treated in three major IVF centres in Bahrain from 2007 until present. All the centres refer their samples to satellite PGD laboratories. The tests were initially focussed on aneuploidy screening for 5 chromosomes by the technique of FISH, which was gradually shifted to 24-chromosome screening by A-CGH in 2013 through 2015 and later substituted with NGS when the technique was made available. The number of cases, the types of PGT tests performed, justifications of testing and the pregnancy outcome are all reviewed, analysed and evaluated. Results At least 70% of cases exhibited an unjustified FISH test, while 30% requested gender selection in favour of male embryos. As for screening and diagnosis of monogenic disorders, about 70% of cases displayed a trait status for hemoglobinopathies which are common in Bahrain (5%) and the eastern province of KSA (4.5%). Lower pregnancy success rates were observed mainly in FISH cases (0-15%), but they showed higher values (17- 48%) in A- CGH (p Conclusions and Recommendations Our health care system in the GCC needs urgent implementation of quality standards to improve the practices of pre- implantation genetic testing. In view of the current bioethical situation of genetic testing in GCC, measures for regulating practises of PGT under a strict controlling system will be suggested for future implementation. Ethical problems in genetic testing of embryoscan be avoided in GCC by considering the following:

Pre-implantation genetic testing (PGT) is a crucial process for selecting embryos created through assisted reproductive technology (ART). Couples with chromosomal rearrangements, infertility, recurrent miscarriages, advanced maternal age, known single-gene disorders, a family history of genetic conditions, previously affected pregnancies, poor embryo quality, or congenital anomalies may be candidates for PGT. Preimplantation genetic testing for aneuploidies (PGT-A) enables the selection and transfer of euploid embryos, significantly enhancing implantation rates in assisted reproduction. Fluorescence in situ hybridization (FISH) is the preferred method for analyzing biopsied cells to identify these abnormalities. While FISH is a well-established method for identifying sperm aneuploidy, NGS offers a more comprehensive assessment of genetic material, potentially enhancing our understanding of male infertility. Chromosomal abnormalities, arising during meiosis, can lead to aneuploid sperm, which may hinder embryo implantation and increase miscarriage rates. This review provides a comparative analysis of fluorescence in situ hybridization (FISH) and next-generation sequencing (NGS) in sperm evaluations, focusing on their implications for preimplantation genetic testing. This analysis explores the strengths and limitations of FISH and NGS, aiming to elucidate their roles in improving ART outcomes and reducing the risk of genetic disorders in offspring. Ultimately, the findings will inform best practices in sperm evaluations and preimplantation genetic testing strategies.