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

Our study established an effective next-generation sequencing (NGS) protocol for four-factor preimplantation genetic testing (PGT) using α- and β-thalassemia, human leukocyte antigen (HLA) typing, and aneuploidy screening. Three couples, in whom both partners were α- and β-double thalassemia carriers, underwent PGT between 2016 and 2018. These individuals sought an opportunity for hematopoietic stem cell transplantation to save their children from β-thalassemia major. A total of 35 biopsied trophectoderm samples underwent multiple displacement amplification (MDA). PGT for α- and β-thalassemia and HLA typing were performed on MDA products using NGS-based single-nucleotide polymorphism (SNP) haplotyping. Although two samples failed MDA, 94.3% (33/35) of samples were successfully amplified, achieving conclusive PGT results. Furthermore, 51.5% (17/33) of the embryos were diagnosed as unaffected non-carriers or carriers. Of the 17 unaffected embryos, nine (52.9%) were tested further and identified as euploid via NGS-based aneuploid screening, in which five had HLA types matching affected children. One family did not achieve any unaffected euploid embryos. The two other families transferred HLA-matched and unaffected euploid embryos, resulting in two healthy ‘savior babies.’ NGS-PGT results were confirmed in prenatal diagnosis. Therefore, NGS-SNP was effective in performing PGT for multipurpose detection within a single PGT cycle.

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.

Study question Do different trophectoderm biopsy techniques affect mosaicism rate in human blastocysts? Summary answer No statistical significance was found between biopsy techniques and mosaicism rate. However, an increase in mosaicism rate was observed when the flicking technique was used. What is known already Mosaicism is defined as two or more distinct cell lines within an embryo. Recent advances in Next Generation Sequencing (NGS) technology with an increased sensitivity allows a higher accuracy in quantification of mosaic levels in biopsied cells. The incidence of mosaicism is widely debated as there are many attributing technical and biological factors. Since, trophectoderm biopsy is a technically challenging process, it is crucial to ensure that the both biopsied cells and blastocyst suffers minimal damage during biopsy. Study design, size, duration This is a prospective study involving 222 patients (age range= 18–44, mean age= 31.5) who underwent IVF cycles in Alpha IVF, Malaysia from March 2019 to August 2019. Six hundred and sixty-eight (668) of the blastocysts were biopsied on Day 5 (Group 1) while 177 blastocysts were biopsied on Day 6 (Group 2). The blastocysts in these groups were further categorised into their corresponding biopsy techniques: (A) laser+pulling; (B) laser+flicking; (C) flicking only. Participants/materials, setting, methods Blastocysts which were at least fair graded (Gardner, 1999) were biopsied and vitrified (Cryotec, Japan). The number of biopsied cells ranged from 5 to 10 cells. All biopsied trophectoderm samples were subjected to Preimplantation Genetic Testing for Aneuploidy (PGT-A) with Next Generation Sequencing (NGS) (Ion Torrent, USA). Chromosomal mosaicism analysis was done using ReproSeq Mosaic PGS w1.1 workflow. Trophectoderm biopsied sample which were tested to have 20% to 80% aneuploid cells were reported as mosaic. Main results and the role of chance In Group 1, the mosaicism rates for biopsy technique A, B and C were 23.3% (104/446), 28.2% (58/206) and 37.5% (6/16) respectively. In Group 2, the mosaicism rates for biopsy technique A, B and C were 14.6% (7/48), 19.5% (23/118) and 27.3% (3/11) respectively. There were no significant differences (p > 0.05) in mosaicism rates between all study groups and subgroups. Limitations, reasons for caution Although no statistical significance was found between trophectoderm biopsy techniques and the prevalence of mosaicism, there is a trend of an increase in mosaicism rate when the flicking technique was used. Therefore, further studies with a larger sample size should be undertaken. Wider implications of the findings: Our study demonstrates a trend in the decrease of mosaicism rate when laser pulses was used to loosen the cell junction of targeted cells. Hence, in place of the flicking method alone, laser pulses should be applied during trophectoderm biopsy if our findings are confirmed in a larger controlled study. Trial registration number Not applicable

Preimplantation Genetic Testing (PGT) enables the genetic analysis of embryos generated through in vitro fertilization (IVF). Traditional applications of PGT include PGT for aneuploides (PGT-A - to identify whole-chromosome aneuploidies), PGT for structural rearrangements (PGT-SR - to detect chromosomal imbalances resulting from a balanced structural rearrangement, such as chromosomal translocations or inversions, in one of the patients), and PGT for monogenic disorders (PGT-M - to minimize the risk of transmitting a known inherited monogenic disorder). Recent advancements in Next Generation Sequencing (NGS) technologies used in PGT, combined with a substantial reduction in associated costs, have made Whole Genome Sequencing (WGS) a feasible approach at the embryonic level. This methodology provides genome-wide data, albeit typically at low coverage depth.These technological developments have enabled novel applications of PGT, such as screening embryos for monogenic variants (including de novo mutations) and polygenic embryo screening (PES). However, these approaches raise significant limitations and challenges, particularly from ethical and genetic counseling perspectives.This session will offer an overview of the key ethical and counseling dilemmas associated with the use of these emerging technologies, in order to facilitate a broader discussion on the scope and boundaries of PGT.

Personalized medicine for the embryo and the fetus - Options in modern genetics influence preconception and prenatal choices.

Hereditary white matter disease is a series of progressive genetic diseases that mainly affect the white matter of the central nervous system. The development of molecular genetics enables the clinical diagnosis, carrier detection, and prenatal diagnosis of hereditary white matter disease. Here, we block the transmission of pathogenic variants in ABCD1 and NOTCH3 in a family with cerebral white matter disease via preimplantation genetic testing (PGT). Pathogenic genes were identified based on clinical manifestations, genetic background, and the results of targeted gene capture sequencing. A blastocyst biopsy was performed, and multiple annealing and looping-based amplification (MALBAC), next-generation sequencing (NGS), and single nucleotide polymorphism (SNP) arrays were used to analyze ploidy and the state of the gene mutations. The proband (III:1) had hemizygous mutations in ABCD1 (c.323C>A (p.Ser108 *) and c.775C>T (p.Arg259Trp)) and heterozygous mutations in NOTCH3 (c.1630C>T (p.Arg544Cys)), which were maternally inherited (II:2). After genetic analysis, a euploid blastocyst without ABCD1 and NOTCH3 variations was transferred. A healthy male baby was born at full term, and the results of prenatal diagnosis by amniocentesis in the second trimester verified the results of PGT. To our knowledge, this is the first report of simultaneously blocking the transmission of pathogenic variants in ABCD1 and NOTCH3 via PGT. This report highlights the feasibility and effectiveness of PGT in preventing cerebral adrenoleukodystrophy (cALD) and cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) and provides valuable insights for the diagnosis and treatment of similar cases.

895 Maternal and neonatal outcomes associated with preimplantation genetic testing in IVF twin pregnancies

EMBRYONIC MOSAICISM IN PREIMPLANTATION GENETIC TESTING (PGT): EVALUATION OF VARIABLES THAT COULD AFFECT ITS FREQUENCY

To what extent does a trophectoderm (TE) biopsy reliably reflect the chromosomal constitution of the inner cell mass (ICM) in human blastocysts? Concordance between TE and ICM was established in 62.1% of the embryos analysed. Next generation sequencing (NGS) platforms have recently been optimised for preimplantation genetic testing for aneuploidies (PGT-A). However, higher sensitivity has led to an increase in reports of chromosomal mosaicism within a single TE biopsy. This has raised substantial controversy surrounding the prevalence of mosaicism in human blastocysts and the clinical implications of heterogeneity between the TE and ICM. To define the distribution and rate of mosaicism in human blastocysts, we assessed chromosomal profiles of the ICM and multiple TE portions obtained from the same embryo. We evaluated donated embryos with an unknown chromosomal profile (n = 34), as well as PGT-A blastocysts, previously diagnosed as abnormal or mosaic (n = 24). Our intra-embryo comparison included a total of 232 samples, obtained from 58 embryos. Four embryo samples, including the ICM and three distinct TE portions, were acquired from good quality blastocysts by micromanipulation. Whole genome amplification (WGA), followed by NGS was performed on all embryo segments. Profiles were compared between samples from the same embryo, while the results from pretested blastocysts were further correlated to the original report. The embryos investigated in our untested group were obtained from good prognosis patients (n = 25), with maternal age ranging from 23 to 39 years. For the pretested embryo group, maternal age ranged from 23 to 40 years (n = 18). We uncover chromosomal mosaicism, involving both numerical and structural aberrations, in up to 37.9% of the blastocysts analysed. Within the untested group, the overall concordance between the ICM and all TE portions was 55.9%. A normal ICM was detected in 20.6% of blastocysts for which at least one TE portion showed a chromosomal aberration. Conversely, 17.6% of embryos presented with mosaic or uniform abnormalities within the ICM, while showing normal or mosaic TE profiles. For the pretested blastocysts, the overall concordance between the ICM and all TE samples was 70.8%. However, 50% of embryos previously diagnosed with mosaicism did not confirm the original diagnosis. Notably, 31.3% of embryos with a mosaic aberration reported in the original TE biopsy, revealed a euploid profile in the ICM and all three TE samples. Taken together, concordance between the ICM and all TE portions was established in 62.1% of blastocysts, across both embryo groups. Finally, we could not observe a significant effect of age on embryo mosaicism (P = 0.101 untested group; P = 0.7309 pretested group). Similarly, ICM and TE quality were not found to affect the occurrence of chromosomal mosaicism (P = 0.718 and P = 0.462 untested group; P = 1.000 and P = 0.2885 pretested group). All data that support the findings of this study are available online in Vivar (http://cmgg.be/vivar) upon request. Evaluating biological variation in some instances remains challenging. The technological limitations of sampling mitotic errors that lead to mosaicism, as well as WGA artefacts, warrant careful interpretation. Our results highlight the complex nature of genetic (in)stability during early ontogenesis and indicate that blastocysts harbour a higher rate of chromosomal mosaicism than may have been anticipated. Moreover, our findings reveal an overall high diagnostic sensitivity and relatively low specificity in the context of PGT-A. This suggests that a considerable proportion of embryos are potentially being classified as clinically unsuitable. Ultimately, more precise quantification will benefit the clinical management of embryo mosaicism. M.P. is supported by the Special Research Fund, Bijzonder Onderzoeksfonds (BOF01D08114). J.T. and L.D. are supported by the agency for innovation through science (131673, 141441). B.H. and this research are supported by the Special Research Fund, Bijzonder Onderzoeksfonds (BOF15/GOA/011). The authors declare no competing interests. Not applicable.

Preimplantation genetic testing (PGT) using Karyomapping is used to screen embryos for single gene disorders prior to implantation. While Karyomapping is not designed to screen for abnormalities in chromosome copy number, this testing is based upon a genome-wide analysis of single nucleotide polymorphisms (SNP) and, as such, some chromosome abnormalities are detected. The aim of this study was to validate whether Karyomapping could provide reliable and accurate PGT for a paternal 46,XY,t(10;19)(p15;p13.3) reciprocal translocation. Feasibility/validation for PGT was performed using DNA from the couple, as well as DNA from the paternal parents and from a previous unbalanced pregnancy. Karyomapping was performed using Illumina's HumanKaryomap-12 BeadChip microarray technology. SNP analysis was performed using BlueFuse Multi software (Illumina). Transmission of the translocation was assessed through the analysis of SNP markers on the chromosome regions of interest. PGT-SR was determined to be feasible as chromosomal SNP analysis could reliably distinguish normal/balanced outcomes from all unbalanced outcomes. The couple transferred a normal/balanced embryo in an elective single embryo transfer procedure following 2 IVF/PGT-SR cycles. A clinical pregnancy was achieved. This is the first report of PGT-SR test validation using Karyomapping for a 46,XY,t(10;19)(p15;p13.3) reciprocal translocation. Karyomapping may offer a means of detecting unbalanced forms of chromosome rearrangements when other PGT platforms fail.

The purpose of this study was to determine the impact of body mass index (BMI) on euploidy rates for in vitro fertilization (IVF) cycles with preimplantation genetic testing (PGT) utilizing primarily next-generation sequencing (NGS). This retrospective cohort study included women aged ≤ 45years who underwent IVF/PGT between September 2013 and September 2020 at a single university-affiliated fertility center. The primary outcome was euploidy rate. Secondary outcomes included peak serum estradiol (E2), number of oocytes retrieved, oocyte maturation rate, high-quality blastulation rate, clinical loss rate (CLR), clinical pregnancy rate (CPR), and ongoing pregnancy/live birth rate (OPR/LBR). The study included 1335 IVF cycles thatwere stratified according toBMI (normal, n = 648; overweight, n = 377; obese, n = 310). The obese group was significantly older with significantly lower baseline FSH, peak E2, high-quality blastulation rate, and number of embryos biopsied than the normal group. Overall euploidy rates were not significantly different between BMI groups (normal 36.4% ± 1.3; overweight 37.3% ± 1.8; obese 32.3% ± 1.8; p = 0.11), which persisted after controlling for covariates (p = 0.82) and after stratification of euploidy rate by age group and by number of oocytes retrieved per age group. There were no significant differences in CLR, CPR, and OPR/LBR across BMI groups. Despite a lower high quality blastulation rate with obesity, there is not a significant difference in euploidy rates across BMI groups in women undergoing IVF/PGT.

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.

Constitutional and Somatic Variants in ARID5B in Pediatric B-Cell Precursor Acute Lymphoblastic Leukemia

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.