Genetic Testing For Monogenic Disease Research Articles

P-512 Combined use of genetic diagnosis and algorithms for embryo quality assessment using artificial intelligence improves selection of embryos leading to clinical pregnancy

Abstract Study question Analyze the results of our genetic diagnosis program, evaluating the morphokinetics of the embryo using an automated algorithm, based on its morphokinetic development and quality. Summary answer Transfers with a higher KIDScore D5 v3.2 euploid embryo have a significantly higher chance of achieving pregnancy. What is known already Embryo selection using morphokinetic algorithms is a technique that allows us to select embryos with better quality and perform more transfers of a single embryo improving the clinical outcomes of treatments, this technique combined with the study of aneuploidies and genetic diseases help us to increase the chances of success in embryo implantation and in the results of live newborn at home. Currently, there are techniques available for their diagnoses that, despite the impact of the processes, allow us to select euploid embryos with greater potential for implantation. Study design, size, duration Retrospective study between 1/5/2022 and 31/12/2023 of patients undergoing preimplantation genetic testing for aneuploidy (PGTA) and preimplantation genetic testing for monogenic diseases (PGTM). A single euploid embryo was transferred. Transferred embryos are compared according to KIDScore D5 v3.2. Participants/materials, setting, methods A total of 171 cycles have been performed with PGT, 97 patients have had at least one euploid embryo or transferable mosaic. The single embryos transferred euploids are compared according to the KIDScore D5 v3.2 automated system that ranks each embryo from one to ten, based on their morphokinetic development and quality. Implantation rate, clinical gestation rate and miscarriage are analyzed. Main results and the role of chance A total of 84 single embryo transfers were performed after PGT with an implantation rate of 60.71% and a clinical gestation rate per transfer of 52.38%. According to the morphokinetic KIDScore D5 v3.2 of the transferred euploid embryo, patients with negative pregnancy had a mean KIDScore D5 v3.2 of 6.5±1.3 and for patients with positive gestation it was 7.5±1.4 (S). The mean maternal age of patients with negative and positive pregnancy was 32.8±3.5 and 34.8±3.9, respectively (NS). For the clinical gestation rate, the mean KIDScore D5 v3.2 was 7.48±1.6 (S). The miscarriage rate was 7.84%. Limitations, reasons for caution The main challenges encompass genetic and reproductive counseling and the most efficient approach to blastocyst biopsy. Wider implications of the findings Our data suggest that transfers with a higher KIDScore D5v3.2 euploid embryo have a significantly higher probability of achieving pregnancy. The evolutionary gestation rate is significantly higher when the morphokinetics of the embryo are better. Further studies with a larger number of cases are needed to better assess this trend. Trial registration number not applicable. non-clinical trials

P-536 Preimplantation Genetic Testing for hereditary breast and ovarian cancer syndrome due to BRCA pathogenic variant : the parisian center experience

Abstract Study question What are the outcomes of Preimplantation genetic testing for monogenic disease (PGT-M) for BRCA pathogenic variants? Summary answer Among couples entering a PGT-M program for BRCA pathogenic variants, 23.5% achieved a live-birth. What is known already BRCA pathogenic variants are associated with an oncologic predisposition with variable penetrance and are accessible to screening and preventive procedures to avoid or early detect the development of cancer. Transmission of BRCAmutations to offspring poses a significant reproductive concern for BRCA mutation carriers. PGT-M offers a solution to prevent this mutation transmission. Despite reproductive challenges related to BRCA status, there are no existing international guidelines for applying PGT-M in these situations. In France, authorization for BRCA related PGT-M is case-specific, considering the personal and familial severity of the disease and submitted to a multidisciplinary prenatal diagnosis center (CPDPN). Study design, size, duration This retrospective observational study, is a single-center analysis of 46 requests for PGT-M related to BRCA pathogenic variants at our PGT center from January 2015 to December 2022. Participants/materials, setting, methods Forty-six couples asked for PGT-M for BRCA pathogenic variant carried by one of the partner. The present study involves the examination of data encompassing referrals, decisions as well as clinical outcomes associated with PGT-M. Main results and the role of chance Forty-six requests for PGT-M due to the risk of hereditary predisposition to breast and ovarian cancer have been received: 36 for a pathogenic variant in the BRCA1 gene and 10 for BRCA2. Nine couples ultimately did not proceed and dropped out. Eight requests were declined, either due to the refusal of the multidisciplinary prenatal diagnosis center (CPDPN) based on family history (n = 4) or alteration of ovarian reserve (n = 4), the 2 latter requests are still under investigation. In total, requests from 27 couples (55%) were considered eligible and feasible. Among them, 17 (63%) underwent at least one PGT-M cycles (5 dropped out before the beginning and 5 are still pending). Overall, 33 ovarian stimulation cycles for in vitro fertilization were initiated, resulting in the retrieval of 192 mature oocytes and the formation of 145 embryos. Of these, 97 were biopsied, and 38 did not carried the BRCA pathogenic variant. A total of 21 fresh or frozen embryo transfers were performed and 8 pregnancies were achieved, leading to 4 deliveries and 5 live births (23.5% of couples who started a PGT-M cycle). Limitations, reasons for caution In the absence of established guidelines, there is variability in information regarding PGT-M among oncogeneticists. Thus, only selected carriers of BRCA pathogenic variants are addressed to our PGT centers. Additionally, women participating in a fresh PGT cycle were selected on several criteria including ovarian reserve parameters. Wider implications of the findings PGT-M provides a viable alternative for couples seeking to prevent the transmission of BRCA mutations. To increase the PGT-M cumulative live birth rate, the transfer of male embryos carrying the variant, assumed to have lower tumor risks, should be considered and discussed with patients. Trial registration number Not applicable

P-540 Strategic implementation of PGT-M for multiple genes post qhole exome sequencing in couples with familial history: a comprehensive study

Abstract Study question What advantages does Whole Exome Sequencing offer to couples with a familial history of hereditary disorders? Summary answer WES offers a comprehensive risk assessment, facilitating the development of an effective PGT-M for Multiple Genes in the prevention of severe diseases in their offspring What is known already PGT-M enables the transfer of unaffected embryos to couples at risk of transmitting monogenic disorders. With the advancements in technologies like Whole Exome Sequencing (WES) and Whole Genome Sequencing (WGS) identifying causal mutations, there is a growing demand for PGT-M applications that concurrently assess multiple genetic disorders. This is particularly crucial, as families initially referred for one condition may later be discovered to be at risk for additional monogenic disorders due to the common carriership status of the couples. Study design, size, duration This is a retrospective study including couples with previous familial history of genetic disorders referred to a single genetic unit between years 2020-2024. Either WES-DUO or WES-TRIO analysis were performed in the absence or presence of an affected child, respectively. Participants/materials, setting, methods WES-DUO or WES-Trio analyses were performed on 94 couples with a familial history of genetic diseases encompassing diverse medical domains. Around 50% of the couples exhibited consanguinity. Utilizing the Novaseq 6000 next-generation sequencing platform, exonic regions were sequenced with an average coverage of 100X. Bioinformatic analysis followed the manufacturer’s recommended workflow, and variant analysis adhered to ACMG criteria, ClinVar, and HGMD. Segregation analysis was conducted through Sanger sequencing using region-specific primers. Main results and the role of chance WES, followed by segregation analysis, pinpointed reportable pathogenic or likely pathogenic variants in 46 out of 94 families as the root cause of the referral condition. Interestingly, 42 of these families exhibited at least one additional common gene mutation in both partners. Notably, among the 27 families with confirmed diagnoses, in vitro fertilization (IVF) and preimplantation genetic testing for monogenic disorders (PGT-M) were undertaken. Within this cohort, PGT-M for multiple genes was successfully conducted in 22 families, 96 embryos were found to be transferable resulting in the birth of healthy infants. Limitations, reasons for caution The overall diagnostic confirmation rate of WES analysis reached 48.9%. A quite high diagnostic yield achieved in this study is notably influenced by the substantial prevalence of consanguineous cases. Wider implications of the findings This study highlights the need for implementing PGT-M for multiple genes. Failure to undergo such testing would potentially lead to the birth of children affected by other conditions.This study also underscores the efficiency of encompassing WES, mutation confirmation, segregation analysis, bioinformatic processing and PGT applications in a single genetic unit. Trial registration number none

P-504 Examining the success rates following a second embryo biopsy for inconclusive results in preimplantation genetic testing for monogenic diseases (PGT-M): a matched case-control study

Abstract Study question Can an embryo re-biopsy for a previous inconclusive result influence clinical pregnancy compared to a successful PGT-M diagnosis from the first biopsy? Summary answer The embryo re-biopsy intervention for PGT-M seems to reduce the implantation potential of a blastocyst. What is known already PGT-M allows embryo genetic profiling prior to transfer in couples with hereditary genetic disorders, as part of in vitro fertilization process. The need of a trophectoderm biopsy for the retrieval of a fragment of about 8-10 blastomeres is generally sufficient for optimal DNA amplification and molecular analysis. However, a proportion of embryos (approximately 10-12%) fails to be properly diagnosed after PGT-M testing due to inconclusive results. Therefore, as an attempt to obtain a genetic diagnosis, the embryo biopsy can be repeated on the same embryos although retesting involves a second round of biopsy and a second round of vitrification. Study design, size, duration Twenty-seven embryos from women undergoing PGT-M at an infertility center and transferred after two biopsies for genetic analysis were matched for age, fertility status, and study period, to embryos transferred after a conclusive PGT-M result after the first biopsy. The main outcome was the clinical pregnancy rate following embryo transfers in cases in whom embryos had a single biopsy, and when a re-biopsy was performed. The secondary outcome was the live birth rate. Participants/materials, setting, methods Ovarian stimulation, oocyte collection and fertilization were performed according to standard protocols. Embryos were cultured to blastocyst stage in groups of up to five, using one-step media. Blastocysts were biopsied on day 5, 6, or 7, employing either the pulling or flicking method based on their appearance. Biopsied cells were stored at -20 °C for genetic analysis. Vitrification was performed using Kitazato protocol. If results were inconclusive, a second biopsy was performed in surviving blastocysts. Main results and the role of chance The median [interquartile range] number of retrieved and injected oocytes was similar between the two groups, as well as fertilization rate and number of blastocysts obtained. No difference between the two groups was also observed pertaining to the day of embryo culture in which the first biopsy was performed. Conversely, the number of non-diagnosticated and transferable blastocysts was lower in patients whose blastocysts were re-biopsied. In 90% of the re-biopsies an informative result was obtained. Clinical pregnancy rate was 52% (95% CI: 34-69) following the transfer of a single-biopsy blastocyst and 30% (95% CI: 16-48) following the transfer of a re-biopsied blastocyst. The likelihood to have a healthy baby was 33% (95% CI: 19-52) following the transfer of a blastocyst biopsied once and 22% (95% CI: 11-41) following the transfer of a re-biopsied blastocyst. Limitations, reasons for caution The matching design, which aligns women’s age and fertility status in the two groups, allowed the assessment of pregnancy rates while avoiding potential confounding factors associated with retrospective analysis. Given that it is the first study with this design, a larger sample size is necessary to further elucidate these findings. Wider implications of the findings Despite the limited sample size, these results are of extreme clinical relevance. They offer valuable insights for guiding decisions about embryos with unclear results, contributing to the improvement of strategies for selecting those with the highest probability of pregnancy. Trial registration number not applicable

P-528 Establishing sequencing coverage depth for preimplantation genetic testing for monogenic disorders (PGT-M) using a specific targeted next generation sequencing (tNGS)

Abstract Study question What is the sufficient sequencing coverage read depth (RD) for direct variant analysis for PGT-M using a specific tNGS assay? Summary answer Downsampling modelling experiments identified a minimum sequencing depth of 50X that is recommended for PGT-M with this specific tNGS assay. What is known already Properly developed and validated multiplex PCR amplification and NGS-based analysis reliably produces PGT-M diagnoses with low allele and amplification failure at RD of hundreds of reads when analyzing a 3-10 cell trophectoderm (TE) biopsy. However, the RD can be affected by amplification efficiency in a specific genomic region and therefore affect the reproducibility of variant detection: the higher the number of aligned reads, the higher the confidence of base calling at a particular position. Downsampling is a powerful bioinformatics tool for modelling comparisons between samples or between replicates, identifying analytical sequencing depth limits for accurate clinical diagnosis. Study design, size, duration This is a retrospective comparative diagnostic test accuracy (DTA) and verification study. Twenty-three embryos from 8 patients that had undergone PGT-M with clinically established Taqman qPCR genotyping were used as positive controls. The primary outcome was to establish the lowest RD point of down sampling where allelic frequency is reliably detected at heterozygosity between 20-80% in known positive controls. A further 30 PGT-M clinical cases were used to verify analytical performances under clinical conditions. Participants/materials, setting, methods Embryo biopsies were pre-amplified with a multiplex primer pool for PGT-M and PGT-A, using a two-step PCR strategy. Sequencing was performed on Illumina NextSeq550. Reads were aligned to a human reference genome (GRCh37/hg19) and processed with an in house bioinformatic pipeline. The RD and alternative allele frequencies (AAF) for PGT-M was calculated. The reads for the heterozygous variants were downsampled to 10X, 20X, 30X, 50X, 100X, 200X, 300X, 500X, and 1000X to establish the cutoff. Main results and the role of chance One or two TE biopsies (N = 44) of 23 embryos that were 100% concordant with previous PGT-M results were tested for 1 to 3 of the 12 variants (MLH1 c.199G>A, RB1 c.1589A>G, SLC16A2 c.604G>A, GNPTAB c.1123C>T, GNPTAB c.3314A>G, WFS1 c.1391C>G, USH2A c.10073G>A, USH2A del exons 5-11, F5 c.1601G>A, GAA c.693-1G>C, GAA c.875A>G, and HEXA c.1274_1277dupTATC). PGT-A analysis of multiple biopsies from the same embryo demonstrated consistent karyotypes (N = 44). The 19 heterozygous variants from 17 biopsies exhibited average RD of 763X (238X -1980X) with average AAF of 50% (44%- 56%). The reads for the heterozygous variants were downsampled to different levels (100,000 replicates). The distribution of the AAF were compared among the different depth levels. At a RD of 50X, the AAF displayed an interquartile range (IQR) of 8% (46%-54%) between the maximum and minimum values from 20% to 78%. Compared to 30X (IQR: 43%-57%, with minimum 13% and maximum 90%), and 100X (IQR: 47%-54%, with minimum 30% and maximum 71%), the RD at 50X generated reliable heterozygous calls with minimum depth requirement. 245 biopsies from 30 clinic cases were analyzed by tNGS-based PGT-M. 90 samples with heterozygous variant calls showed average RD of 979X (57X-5803X) with average AAF of 47% (29%-585). Limitations, reasons for caution The 50X cutoff is based on the heterozygous variants with theoretical 50% AFF and is specific for this PGT-A assay. This evaluation should be corroborated for complicated variants with homologous genes, which might require different thresholds. The aneuploidy at specific chromosome can cause the deviation of AFF from 50%. Wider implications of the findings This specific tNGS-based PGT-M can provide direct variant analysis at a 50X read depth with a high level of consistency compared to established methodologies while allowing for simultaneous aneuploidy screening using the same biopsy and procedural optimization. Trial registration number not applicable

A heatmap for expected cumulative live birth rate in preimplantation genetic testing for monogenic disorders and chromosomal structural rearrangements.

Our objective is to predict the cumulative live birth rate (CLBR) and identify the specific subset within the population undergoing preimplantation genetic testing for monogenic disorders (PGT-M) and chromosomal structural rearrangements (PGT-SR) which is likely to exhibit a diminished expected CLBR based on various patient demographics. We performed a single-centre retrospective cohort study including 1522 women undergoing 3130 PGT cycles at a referral centre for PGT. A logistic regression analysis was performed to predict the CLBR per ovarian stimulation in women undergoing PGT-M by polymerase chain reaction (PCR) or single-nucleotide polymorphism (SNP) array, and in women undergoing PGT-SR by SNP array, array comparative genomic hybridization (CGH) or next-generation sequencing (NGS). The mean age of women was 32.6years, with a mean AMH of 2.75µg/L. Female age and AMH significantly affected the expected CLBR irrespective of the inheritance mode or PGT technology. An expected CLBR < 10% was reached above the age of 42years and AMH ≤ 1.25µg/L. We found no significant difference in outcome per ovarian stimulation between the different PGT technologies, i.e. PCR, SNP array, array CGH and NGS. Whereas per embryo transfer, we noticed a significantly higher probability of live birth when SNP array, array CGH and NGS were used as compared to PCR. In a PGT-setting, couples with an unfavourable female age and AMH should be informed of the prognosis to allow other reproductive choices. The heatmap produced in this study can be used as a visual tool for PGT couples.

The impact of a second embryo biopsy for preimplantation genetic testing for monogenic diseases (PGT-M) with inconclusive results on pregnancy potential: results from a matched case-control study.

To evaluate whether a second biopsy, following a first diagnostic failure on blastocysts tested for preimplantation genetic testing for monogenic diseases (PGT-M), allows to obtain genetic diagnosis and to what extent this procedure can influence clinical pregnancy and live birth rates compared to the PGT-M process with a successful genetic diagnosis from the first biopsy. Embryos from women who underwent PGT-M in an infertility centre and who had been transferred after two biopsies for genetic analysis (n = 27) were matched in a 1:1 ratio accordingly to women's age (± 1year) and fertility status (fertilevsinfertile), as well as with the study period, with embryos who were transferred after receiving a conclusive PGT result straight after the first biopsy (n = 27). The main evaluated outcome was clinical pregnancy rate following embryo transfers in which healthy embryos were transferred after only one biopsy and those in which an embryo was transferred after being re-biopsied. Live birth rate was the secondary outcome. Clinical pregnancy rate was 52% (95% CI: 34-69) following the transfer of a single-biopsy blastocyst and 30% (95% CI: 16-48) following the transfer of a re-biopsied blastocyst. The likelihood to have a healthy baby was 33% (95% CI: 19-52) following the transfer of a blastocyst biopsied once and 22% (95% CI: 11-41) following the transfer of a re-biopsied blastocyst. The re-biopsy intervention seems to considerably reduce the pregnancy potential of a blastocyst. However, a greater sample size is necessary to clarify this issue definitively.

Preimplantation genetic testing for monogenic disorders (PGT-M) offers an alternative strategy to prevent children from being born with hereditary neurological diseases or metabolic diseases dominated by nervous system phenotypes: a retrospective study.

Preimplantation genetic testing for monogenic disorders (PGT-M) is now widely used as an effective strategy to prevent various monogenic or chromosomal diseases. In this retrospective study, couples with a family history of hereditary neurological diseases or metabolic diseases dominated by nervous system phenotypes and/or carrying the pathogenic genes underwent PGT-M to prevent children from inheriting disease-causing gene mutations from their parents and developing known genetic diseases. After PGT-M, unaffected (i.e., normal) embryos after genetic detection were transferred into the uterus of their corresponding mothers. A total of 43 carrier couples with the following hereditary neurological diseasesor metabolic diseases dominated by nervous system phenotypesunderwent PGT-M: Duchenne muscular dystrophy (13 families); methylmalonic acidemia (7 families); spinal muscular atrophy (5 families); infantile neuroaxonal dystrophyand intellectual developmental disorder(3 families each); Cockayne syndrome(2 families); Menkes disease, spinocerebellar ataxia, glycine encephalopathy with epilepsy,Charcot-Marie-Tooth disease, mucopolysaccharidosis, Aicardi-Goutieres syndrome, adrenoleukodystrophy, phenylketonuria, amyotrophic lateral sclerosis, and Dravet syndrome (1 family each). After 53 PGT-M cycles,the final transferable embryo rate was 12.45%, the clinical pregnancy rate was 74.19%, and the live birth rate was 89.47%;a total of 18 unaffected (i.e., healthy) children were born to these families. This study highlights the importance of PGT-M in preventing children born with hereditary neurological diseases or metabolic diseases dominated by nervous system phenotypes.

Proband-independent haplotyping based on NGS-based long-read sequencing for detecting pathogenic variant carrier status in preimplantation genetic testing for monogenic diseases.

Preimplantation genetic testing for monogenic diseases (PGT-M) can be used to select embryos that do not develop disease phenotypes or carry disease-causing genes for implantation into the mother's uterus, to block disease transmission to the offspring, and to increase the birth rate of healthy newborns. However, the traditional PGT-M technique has some limitations, such as its time consumption, experimental procedural complexity, and the need for a complete family or reference embryo to construct the haplotype. In this study, proband-independent haplotyping based on NGS-based long-read sequencing (Phbol-seq) was used to effectively construct haplotypes. By targeting the mutation sites of single gene disease point mutations and small fragment deletion carriers, embryos carrying parental disease-causing mutations were successfully identified by linkage analysis. The efficiency of embryo resolution was then verified by classical Sanger sequencing, and it was confirmed that the construction of haplotype and SNP linkage analysis by Phbol-seq could accurately and effectively detect whether embryos carried parental pathogenic mutations. After the embryos confirmed to be nonpathogenic by Phbol-seq-based PGT-M and confirmed to have normal copy number variation by Phbol-seq-based PGT-A were transplanted into the uterus, gene detection in amniotic fluid of the implanted embryos was performed, and the results confirmed that Phbol-seq technology could accurately distinguish normal genotype embryos from genetically modified carrier embryos. Our results suggest that Phbol-seq is an effective strategy for accurately locating mutation sites and accurately distinguishing between embryos that inherit disease-causing genes and normal embryos that do not. This is critical for Phbol-seq-based PGT-M and could help more single-gene disease carriers with incomplete families, de novo mutations or suspected germline mosaicism to have healthy babies with normal phenotypes. It also helps to reduce the transmission of monogenic genetic diseases in the population.

Preimplantation genetic testing as a means of preventing hereditary congenital myasthenic syndrome caused by RAPSN.

Congenital myasthenic syndrome is a heterogeneous group of inherited neuromuscular transmission disorders. Variants in RAPSN are a common cause of CMS, accounting for approximately 14%-27% of all CMS cases. Whether preimplantation genetic testing for monogenic disease (PGT-M) could be used to prevent the potential birth of CMS-affected children is unclear. Application of WES (whole-exome sequencing) for carrier testing and guidance for the PGT-M in the absence of a genetically characterized index patient as well as assisted reproductive technology were employed to prevent the occurrence of birth defects in subsequent pregnancy. The clinical phenotypes of stillborn fetuses were also assessed. The family carried two likely pathogenic variants in RAPSN(NM_005055.5): c.133G>A (p.V45M) and c.280G>A (p.E94K). And the potential birth of CMS-affected child was successfully prevented, allowing the family to have offspring devoid of disease-associated variants and exhibiting a normal phenotype. This report constitutes the first documented case of achieving a CMS-free offspring through PGT-M in a CMS-affected family. By broadening the known variant spectrum of RAPSN in the Chinese population, our findings underscore the feasibility and effectiveness of PGT-M for preventing CMS, offering valuable insights for similarly affected families.

Serum uric acid did not affect embryonic and pregnancy outcomes in women without PCOS during IVF procedures.

Serum uric acid (UA) levels are associated with many systemic diseases. A previous study confirmed the association between high serum uric acid levels and poor prognosis of in vitro fertilization (IVF) treatment in polycystic ovary syndrome (PCOS) patients. This study aimed to explore the correlation between serum uric acid levels and reproductive outcomes in patients without PCOS. A retrospective study that included 1057 patients who underwent pre-implantation genetic testing for monogenic disorders (PGT-M) treatment from January 2013 to December 2020 was conducted. The study population was further divided into 3 groups according to serum UA levels: the ≤250 μmol/L group, the 251-360 μmol/L group, and the >360 μmol/L group. The controlled ovarian hyperstimulation (COH) treatment outcomes, embryonic treatment outcomes and pregnancy outcomes of the first frozen embryo transfer (FET) cycle were compared among groups. Multivariable linear regression and binary regression were applied to detect the association between IVF outcomes and serum uric acid levels. The number of retrieved oocytes, fertilization rate, viable embryo rate, blastocyst formation rate and euploid rate were not associated with serum uric acid levels. The mature oocyte rate was negatively correlated with serum uric acid levels. The pregnancy outcomes of the first FET cycle were also not associated with serum uric acid levels. After adjustment for BMI, the perinatal outcomes were not associated with serum uric acid levels. IVF treatment outcomes were not associated with serum uric acid levels in patients without PCOS.

Development of preimplantation genetic testing for monogenic reference materials using next-generation sequencing

ObjectivePreimplantation genetic testing for monogenic disorders (PGT-M) has been used for over 20 years to detect many serious genetic conditions. However, there is still a lack of reference materials (RMs) to validate the test performance during the development and quality control of PGT-M.MethodSixteen thalassemia cell lines from four thalassemia families were selected to establish the RMs. Each family consisted of parents with heterozygous mutations for α- and/or β-thalassemia and two children, at least one of whom carried a homozygous thalassemia mutation (proband). The RM panel consisted of 12 DNA samples (parents and probands in 4 families) and 4 simulated embryos (cell lines constructed from blood samples from the four nonproband children). Four accredited genetics laboratories that offer verification of thalassemia samples were invited to evaluate the performance of the RM panel. Furthermore, the stability of the RMs was determined by testing after freeze‒thaw cycles and long-term storage.ResultsPGT-M reference materials containing 12 genome DNA (gDNA) reference materials and 4 simulated embryo reference materials for thalassemia testing were successfully established. Next-generation sequencing was performed on the samples. The genotypes and haplotypes of all 16 PGT-M reference materials were concordant across the four labs, which used various testing workflows. These well-characterized PGT-M reference materials retained their stability even after 3 years of storage.ConclusionThe establishment of PGT-M reference materials for thalassemia will help with the standardization and accuracy of PGT-M in clinical use.

A clinical predictive model for live birth in women of advanced age undergoing PGT cycles.

The trend of delaying childbirth has resulted in a growing number of advanced-aged women who are opting for preimplantation genetic testing (PGT) to screen for monogenic diseases or structural chromosomal rearrangements (PGT-M and PGT-SR). This increase in demand necessitates the development of a clinical predictive model for live birth outcomes in these women. Therefore, the objective of this study is to construct a comprehensive predictive model that assesses the likelihood of achieving a successful live birth in advanced-aged women undergoing PGT-M and PGT-SR treatments. A retrospective cohort study of 37-45-year-old women undergoing preimplantation genetic testing for monogenic disease or structural chromosomal rearrangement cycles from 2010 to 2021 was conducted at a university hospital reproductive centre. The purpose was to develop a clinical predictive model for live birth in these women. The main outcome studied was the cumulative live birth rate in the first or subsequent cycles. Developing a decision tree enabled a comprehensive study of clinical parameters and expected outcomes. The analysis included 158 women undergoing 753 preimplantation genetic testing cycles. The cumulative live birth rate was 37.342% (59/158). Decision tree analysis revealed that women aged ≤ 40.1 or women > 40.1 with one or more top-quality transferable embryos in their first cycle had the best chance for a live baby (56% and 41%, respectively). Those older than 40.1 without top-quality embryos and seven or fewer dominant follicles had no live births. A Kaplan-Meier curve showed that for autosomal dominant diseases, there was a negligible increase in live birth rate after three cycles, compared to six cycles in autosomal recessive inheritance. In older women, the chance of delivering after repeated cycles is higher in those with at least one top-quality unaffected embryo in their first preimplantation genetic testing cycle. Additional preimplantation genetic testing cycles after three in carriers of an autosomal dominant disorder and six in those with an autosomal recessive disorder should be considered prudently.

PGT-M for Premature Ovarian Failure Related to CGG Repeat Expansion of the FMR1 Gene.

Primary ovarian failure (POF) is caused by follicle exhaustion and is associated with menstrual irregularities and elevated gonadotropin levels, which lead to infertility before the age of 40 years. The etiology of POI is mostly unknown, but a heterogeneous genetic and familial background can be identified in a subset of cases. Abnormalities in the fragile X mental retardation 1 gene (FMR1) are among the most prevalent monogenic causes of POI. These abnormalities are caused by the expansion of an unstable CGG repeat in the 5' untranslated region of FMR1. Expansions over 200 repeats cause fragile X syndrome (FXS), whereas expansions between 55 and 200 CGG repeats, which are defined as a fragile X premutation, have been associated with premature ovarian failure type 1 (POF1) in heterozygous females. Preimplantation genetic testing for monogenic diseases (PGT-M) can be proposed when the female carries a premutation or a full mutation. In this narrative review, we aim to recapitulate the clinical and molecular features of POF1 and their implications in the context of PGT-M.

First preimplantation genetic testing case of Meckel syndrome with a novel homozygous TXNDC15 variant in a non-consanguineous Chinese family.

Meckel-Gruber syndrome (MKS) is a perinatally lethal, genetically heterogeneous, autosomal recessive condition caused by defective primary cilium formation. So far, the association of TXNDC15-related MKS has been reported in only five independent families from diverse ethnic origins, including Saudi, Pakistani, Estonian, and Indian. Here, we report a fetus diagnosed with MKS at 12 weeks, exhibiting typical ultrasound findings. Low-coverage whole-genome sequencing was used to identify chromosomal abnormalities. Trio-base whole exome sequencing (trio-WES) was performed to investigate the potential pathogenic variants associated with MKS. Preimplantation genetic testing for monogenic disorders (PGT-M) was applied to prevent the transmission of the pathogenic variant. A novel homozygous pathogenic variant in the TXNDC15 gene was identified through trio-WES. The application of PGT-M successfully prevented the transmission of the pathogenic variant and resulted in an ongoing pregnancy. This is the first report of a TXNDC15 variant in the Chinese population and the first PGT case of TXNDC15-related MKS worldwide. The successful application of PGT-M in this family provides a potential approach for other monogenic diseases. Our case expands the variant spectrum of TXNDC15 and contributes to the molecular diagnosis and genetic counseling for MKS. This case underscores the importance of appropriate genetic testing methods and accurate genetic counseling in the diagnosis of rare monogenic diseases.

OneGene PGT: comprehensive preimplantation genetic testing method utilizing next-generation sequencing.

Preimplantation genetic testing for monogenic disorders (PGT-M) allows early diagnosis in embryos conceived in vitro. PGT-M helps to prevent known genetic disorders in affected families and ensures that pathogenic variants in the male or female partner are not passed on to offspring. The trend in genetic testing of embryos is to provide a comprehensive platform that enables robust and reliable testing for the causal pathogenic variant(s), as well as chromosomal abnormalities that commonly occur in embryos. In this study, we describe PGT protocol that allows direct mutation testing, haplotyping, and aneuploidy screening. Described PGT protocol called OneGene PGT allows direct mutation testing, haplotyping, and aneuploidy screening using next-generation sequencing (NGS). Whole genome amplification product is combined with multiplex PCR used for SNP enrichment. Dedicated bioinformatic tool enables mapping, genotype calling, and haplotyping of informative SNP markers. A commercial software was used for aneuploidy calling. OneGenePGT has been implemented for seven of the most common monogenic disorders, representing approximately 30% of all PGT-M indications at our IVF centre. The technique has been thoroughly validated, focusing on direct pathogenic variant testing, haplotype identification, and chromosome abnormality detection. Validation results show full concordance with Sanger sequencing and karyomapping, which were used as reference methods. OneGene PGT is a comprehensive, robust, and cost-effective method that can be established for any gene of interest. The technique is particularly suitable for common monogenic diseases, which can be performed based on a universal laboratory protocol without the need for set-up or pre-testing.

#270 : Single-Center Retrospective Cohort Analysis: Preimplantation Genetic Testing for Monogenic Disorders (PGT-M) for Monogenic Nephropathy

Background and Aims: Hereditary nephropathy, including monogenic nephropathy, is an important cause of renal insufficiency and end-stage renal disease. Therefore, genetic blockade is necessary for couples with monogenic nephropathy. Following routine application of prenatal diagnosis, preimplantation genetic testing for monogenic disorders (PGT-M) has been applied to the genetic blockade of monogenic disease patients. The purpose of this study is to retrospectively analyze genetic counseling process for patients with nephropathy-related disease and provide clinical overviews of patients with monogenic nephropathy who underwent PGT-M. Method: A single-center retrospective cohort study was conducted at Center for Reproductive Medicine, Shandong University from January 2014 to December 2022. 352 couples with nephropathy-related disease were included in the cohort totally. Statistical analysis was performed using Statistical Package for Social Science software 26.0. Results: Of the 352 couples with nephropathy-related disease, 291 underwent genetic counseling, followed by 180 accepting genetic screening. 104 couples with monogenic nephropathy indications proceeded with PGT-M, including 91 of autosomal dominant inheritance, 9 of autosomal recessive inheritance, 3 of X-linked dominant inheritance, and one of X-linked recessive inheritance. 498 blastocysts were tested by PGT-M combined with preimplantation genetic testing for aneuploidy (PGT-A), making 72 transferable embryos, 249 non-transferable embryos, 72 genetic counseling embryos and 7 amplification failedembryos. Finally, 80 vitrified-thawed single blastocyst transfer cycles were performed. Live births occurred in 38 women, of which 37 transferred embryos with non-pathogenic genotypes. The results of prenatal diagnosis of all newborns were consistent with the testing results for embryos transferred. The cumulative live birth, biochemical pregnancy, clinical pregnancy, ongoing pregnancy and pregnancy loss rates were 36.54%, 56.73%, 49.04%, 7.69%, and 22.03% respectively. Conclusion: PGT-M is an effective means of genetic blockade for couples with monogenic nephropathy. The absence of genetic abnormalities detected by prenatal diagnosis in healthy newborns without monogenic nephropathy also underscore its validity.

Public Awareness and Acceptability of PGT-M in Cancer Predisposition Syndromes.

Cancer Predisposition Syndromes (CPSs), also known as Hereditary Cancer Syndromes (HCSs), represent a group of genetic disorders associated with an increased lifetime risk of developing cancer. In this article, we provide an overview of the reproductive options for patients diagnosed with CPS, focusing on the emerging role of Preimplantation Genetic Testing for Monogenic disorders (PGT-M). Specifically, we conducted a literature review about the awareness and acceptability of its application to CPSs. Based on the available data, the awareness of the applicability of PGT-M for CPSs appears to be limited among both patients and physicians, and a heterogeneous set of factors seems to influence the acceptability of the procedure. Our findings highlight the need for increasing education about the use of PGT-M for CPSs. In this context, guidelines developed by professional or institutional bodies would represent a useful reference tool to assist healthcare professionals in providing proper preconception counseling.

State of the Science and Ethical Considerations for Preimplantation Genetic Testing for Monogenic Cystic Kidney Diseases and Ciliopathies.

There is a broad phenotypic spectrum of monogenic polycystic kidney diseases (PKDs). These disorders often involve cilia-related genes and lead to the development of fluid-filled cysts and eventual kidney function decline and failure. Preimplantation genetic testing for monogenic (PGT-M) disorders has moved into the clinical realm. It allows prospective parents to avoid passing on heritable diseases to their children, including monogenic PKD. The PGT-M process involves embryo generation through in vitro fertilization, with subsequent testing of embryos and selective transfer of those that do not harbor the specific disease-causing variant(s). There is a growing body of literature supporting the success of PGT-M for autosomal-dominant and autosomal-recessive PKD, although with important technical limitations in some cases. This technology can be applied to many other types of monogenic PKD and ciliopathies despite the lack of existing reports in the literature. PGT-M for monogenic PKD, like other forms of assisted reproductive technology, raises important ethical questions. When considering PGT-M for kidney diseases, as well as the potential to avoid disease in future generations, there are regulatory and ethical considerations. These include limited government regulation and unstandardized consent processes, potential technical errors, high cost and equity concerns, risks associated with pregnancy for mothers with kidney disease, and the impact on all involved in the process, including the children who were made possible with this technology.

Comprehensive application of multiple molecular diagnostic techniques in pre-implantation genetic testing for monogenic.

Pre-implantation genetic testing for monogenic disorders (PGT-M) is an effective approach to reducing the incidence of birth defects by preventing the transmission of inherited diseases to offspring. However, there are still controversies regarding the detection methods and transplantation of embryos. This paper aims to evaluate the effectiveness of different detection technologies applied to PGT-M through a retrospective analysis of clinical detection data. The carrier status of pathogenic mutations and chromosomal copy number variants (CNVs) in 892 embryos was characterized using next-generation sequencing (NGS), single-nucleotide polymorphism (SNP) array, and PCR-based detection technologies. Clinical data from PGT-M cases were retrospectively analyzed to assess the effectiveness of these detection methods in identifying genetic abnormalities in embryos. A total of 829 embryos were analyzed, with 63 being unsuccessful. Our study revealed that the success rate of detecting deletional mutations using Gap-PCR 84.9%, which is lower than that of SNP array (98.7%) and NGS (92.5%). However, no significant difference was observed when detecting point mutations using any of the methods. These findings suggest that, when detecting deletional mutations, SNP array and NGS are more suitable choices compared to Gap-PCR. While SNP array may have a lower resolution and success rate (80.5%) in analyzing CNVs compared to NGS (95.5%), it may still be useful for revealing certain abnormal types. In conclusion, this study found that SNP analysis is advantageous for identifying polygenic and deletional mutations, whereas NGS is more cost-efficient for detecting common monogenic diseases. Additionally, SNP-based haplotyping and PCR-based direct detection of mutations can be used together to enhance the accuracy and success rates of PGT-M. Our findings offer valuable insights for PGT technicians in choosing suitable detection methods for patients.