Genetic Testing For Monogenic Disease Research Articles

Development and clinical application of a preimplantation genetic testing for monogenic disease (PGT-M) for beta thalassemia in Vietnam

PurposeThe purpose of this research is to study the clinical outcomes using a next-generation sequencing-based protocol allowing for simultaneous testing of mutations in the beta thalassemia (HBB) gene, including single nucleotide polymorphism (SNP) markers for PGT-M along with low-pass whole genome analysis of chromosome aneuploidies for PGT-A.MethodsA combined PGT-M (thalassemia) plus PGT-A system was developed for patients undergoing IVF in Vietnam. Here we developed a system for testing numerous thalassemia mutations plus SNP-based testing for backup mutation analysis and contamination control using next-generation sequencing (NGS). Low -pass next-generation sequencing was used to assess aneuploidy in some of the clinical PGT cases. Patients underwent IVF followed by embryo biopsy at the blastocyst stage for combined PGT-A/M.ResultsTwo cases have completed the entire process including transfer of embryos, while a further nine cases have completed the IVF and PGT-M/A analysis but have not completed embryo transfer. In the two cases with embryo transfer, both patients achieved pregnancy with an unaffected, euploid embryo confirmed through prenatal diagnosis. In the further nine cases, 39 embryos were biopsied and all passed QC for amplification. There were 8 unaffected embryos, 31 carrier embryos, and 11 affected embryos. A subset of 24 embryos also had PGT-A analysis with 22 euploid embryos and 2 aneuploid embryos.ConclusionsHere we report the development and clinical application of a combined PGT-M for HBB and PGT-A for gross chromosome aneuploidies from 11 patients with detailed laboratory findings along with 2 cases that have completed embryo transfer.

Obstetric and Perinatal Outcomes in Pregnancies Conceived After Preimplantation Genetic Testing for Monogenetic Diseases.

To investigate whether the addition of embryo biopsy performed during preimplantation genetic testing for monogenic diseases is associated with a higher risk of obstetric and neonatal complications compared with in vitro fertilization (IVF) without preimplantation genetic testing or spontaneously conceived pregnancies. This is a cohort study of all pregnancies conceived after preimplantation genetic testing for monogenic diseases (PGT-M group) from 2006 to 2018 at Sheba Medical Center, Israel. The control groups included patients who had conceived spontaneously (spontaneous conception group) or by IVF without preimplantation genetic testing (IVF group) and delivered at Sheba Medical Center. The obstetrics outcomes were compared among the groups. Multivariable regression modeling was performed, focusing on the relationship between preimplantation genetic testing and adverse outcomes. Final analysis included 345 singleton and 76 twin deliveries in the PGT-M group. The spontaneous conception group included 5,290 singleton and 92 twin deliveries. The IVF group included 422 singleton and 101 twin deliveries. Among singleton pregnancies, patients in the PGT-M group had a higher rate of hypertensive disorders (6.9%) compared with those in the spontaneous conception group (2.3%; odds ratio [OR] 3.3; 95% CI 1.9-4.8; adjusted odds ratio [aOR] 14.8; 95% CI 7.4-29.8) and the IVF group (4.7%; OR 1.5; 95% CI 0.8-2.7; aOR 5.9; 95% CI 1.9-18.2). Likewise, patients in the PGT-M group had a higher rate of small-for-gestational age neonates (12.4%) compared with those in the spontaneous conception group (3.9%; OR 3.4; 95% CI 2.4-4.9; aOR 2.3; 95% CI 1.5-3.4) and the IVF group (4.5%; OR 3; 95% CI 1.7-5.2; aOR 2.5; 95% CI 1.7-5.2). Among twin pregnancies, patients in the PGT-M group also had an increased rate of hypertensive disorders compared with those in the spontaneous conception group (4.3%; OR 4.1; 95% CI 1.2-13.3; aOR 10.9; 95% CI 2.3-50) and the IVF group (4%; OR 4.5; 95% CI 1.4-14.7; aOR 3.7; 95% CI 1.1-12.8). Pregnancies conceived after preimplantation genetic testing for monogenic disorders were associated with an increased risk of obstetric complications compared with pregnancies conceived spontaneously or by IVF without preimplantation genetic testing.

DOES INTRACYTOPLASMIC SPERM INJECTION (ICSI) PROVIDE ANY BENEFIT OVER IN VITRO FERTILIZATION (IVF) ON PREGNANCY OUTCOMES IN NON-MALE FACTOR INFERTILITY CYCLES UNDERGOING PRE-IMPLANTATION GENETIC TESTING FOR ANEUPLOIDY (PGT-A)?

ICSI is currently the recommended fertilization method for cycles undergoing PGT-A 1. To our knowledge there are no studies evaluating pregnancy outcomes of PGT-A transferred embryos inseminated by IVF versus ICSI. Our objective is to evaluate whether significant differences exist in pregnancy outcomes from PGT-A transferred embryos fertilized by IVF compared to ICSI. Retrospective cohort study at a single academic institution. All frozen embryo transfers (FET) performed from 1/2016 to 2/2020 that underwent PGT-A from trophectoderm biopsy and tested by next generation sequencing were evaluated. Exclusion criteria included diagnosis of male factor infertility, embryos tested for pre-implantation genetic testing for monogenic disorders, structural rearrangements, or HLA-typing, cryopreserved oocytes, blastomere or polar body biopsy, in vitro maturation, rescue ICSI, and split IVF/ICSI cycles. Patient demographics, infertility diagnoses, cycle characteristics, and pregnancy outcomes including not pregnant, biochemical pregnancy, miscarriage, ongoing pregnancy (OP), and live birth (LB) were collected. Primary outcome was OP/LB rates. Secondary outcomes included miscarriage and biochemical rates. Chi-square or Fisher’s exact test, as appropriate, and Mann-Whiney test were used for categorical and continuous variables, respectively. Outcomes between both groups were compared using a repeated-measures mixed-effects logistic regression model, in which the rates were adjusted for confounding variables attained during the univariate screen. Results were considered statistically significant with a p-value <0.05. A total of 317 FET’s met criteria (IVF-150 [47.3%]; ICSI-167 [52.7%]). Significant difference between the IVF and ICSI cohort existed in age (35.8 y/o vs. 36.8 y/o, respectively, p=0.03), BMI (23.6 vs 25.1, respectively, p=0.005), and ethnicities. LB/OP rates and miscarriage/biochemical rates demonstrated no significant differences in IVF vs. ICSI inseminated groups (Table 1). There were no significant differences in pregnancy outcomes in FET’s between IVF and ICSI inseminated embryos that underwent PGT-A. The use of ICSI in non-male factor infertility cycles undergoing PGT-A does not provide an advantage for LB/OP rates over IVF.Table 1IVF (150)ICSI (167)p-valueNot pregnant35.7%37.3%0.79Biochemical12.1%14.8%0.62Miscarriage11.4%7.8%0.43Live birth37.3%35.0%0.69Biochemical/Miscarriage23.6%22.5%0.85Live birth/Ongoing39.8%38.3%0.74 Open table in a new tab

Haplotyping by linked-read sequencing (HLRS) of the genetic disease carriers for preimplantation genetic testing without a proband or relatives

BackgroundIn order to mitigate the risk of allele dropout (ADO) and ensure the accuracy of preimplantation genetic testing for monogenic disease (PGT-M), it is necessary to construct parental haplotypes. Typically, haplotype resolution is obtained by genotyping multiple polymorphic markers in both parents and a proband or a relative. Sometimes, single sperm typing, or tests on the polar bodies may also be useful. Nevertheless, this process is time-consuming. At present, there was no simple linkage analysis strategy for patients without affected relatives.MethodTo solve this problem, we established a haplotyping by linked-read sequencing (HLRS) method without the requirement for additional relatives. First, the haplotype of the genetic disease carriers in the family was constructed by linked-read sequencing, and then the informative single nucleotide polymorphisms (SNPs) in upstream and downstream mutation region were selected to construct the embryo haplotype and to determine whether the embryo was carrying the mutation. Two families were selected to validate this method; one with alpha thalassemia and the other with NDP gene disorder.ResultsThe haplotyping by linked-read sequencing (HLRS) method was successfully applied to construct parental haplotypes without recruiting additional family members; the method was also validated for PGT-M. The mutation carriers in these families were sequenced by linked-read sequencing, and their haplotypes were successfully phased. Adjacent SNPs of the mutation gene were identified. The informative SNPs were chosen for linkage analyses to identify the carrier embryos. For the alpha thalassemia family, a normal blastocyst was transferred to the uterus and the accuracy of PGT-M was confirmed by amniocentesis at 16 weeks of gestation.ConclusionsOur results suggest that HLRS can be applied for PGT-M of monogenic disorders or de novo mutations where the mutations haplotype cannot be determined due to absence of affected relatives.

Timing day-3 vitrification for PGT-M embryos: pre- or post-blastomere biopsy?

To assess the efficacy and clinical outcomes of preimplantation genetic testing for monogenic diseases (PGT-M), following blastomere biopsy prior or following vitrification. A cohort-historical study of all consecutive patients admitted to IVF in a large tertiary center for PGT-M and PCR cycle from September 2016 to March 2020. Patients were divided into 4 groups: Group A1 consisted of patients undergoing day-3 embryos biopsy followed by a fresh transfer of unaffected embryos. Group A2 consisted of Group A1 patients that their surplus unaffected embryos were vitrified, thawed, and transferred in a subsequent FET cycle. Group B1 consisted of patients that their day-3 embryos were vitrified intact (without biopsy) for a subsequent FET cycle. Later embryos were thawed and underwent blastomere biopsies, and the unaffected embryos were transferred, while the surplus unaffected embryos were re-vitrified for a subsequent FET cycle. Group B2 consisted of Group B1 patients that their surplus unaffected embryos were re-vitrified, thawed, and transferred in a subsequent FET cycle. The laboratory data and clinical results were collected and compared between groups. A total of 368 patients underwent 529 PGT-M cycles in our center: 347 with day-3 embryos biopsied before undergoing vitrification (Group A1) and 182 following vitrification and thawing (Group B1). There were no between group differences in embryo survival rate post-thawing, nor the ongoing implantation and pregnancy rates. In PGT-M cycles, the timing of embryos vitrification, whether prior or following blastomere biopsy, has no detrimental effect on post-thawing embryo survival rate, nor their potential ongoing implantation and pregnancy rates.

Complex preimplantation genetic tests for Robertsonian translocation, HLA, and X-linked hyper IgM syndrome caused by a novel mutation of CD40LG gene.

To perform complex preimplantation genetic tests (PGT) for aneuploidy screening, Robertsonian translocation, HLA-matching, and X-linked hyper IgM syndrome (XHIGM) caused by a novel mutation c.156 G>T of CD40LG gene. Reverse transcription PCR (RT-PCR) and Sanger sequencing were carried out to confirm the causative variant of CD40LG gene in the proband and parents. Day 5 and D6 blastocysts, obtained by in vitro fertilization (IVF) with intracytoplasmic sperm injection, underwent trophectoderm (TE) biopsy and whole genomic amplification (WGA) and next generation sequencing (NGS)-based PGT to detect the presence of a maternal CD40LG mutation, aneuploidy, Robertsonian translocation carrier, and human leukocyte antigen (HLA) haplotype. Sanger sequencing data of the genomic DNA showed that the proband has a hemizygous variant of c. 156 G>T in the CD40LG gene, while his mother has a heterozygous variant at the same position. Complementary DNA (cDNA) of CD40LG amplification and sequencing displayed that no cDNA of CD40LG was found in proband, while only wild-type cDNA of CD40LG was amplified in the mother. PGT results showed that only one of the six tested embryos is free of the variant c.156 G>T and aneuploidy and having the consistent HLA type as the proband. Meanwhile, the embryo is a Robertsonian translocation carrier. The embryo was transplanted into the mother's uterus. Amniotic fluid testing results are consistent with that of PGT. A healthy baby girl was delivered, and the peripheral blood testing data was also consistent with the testing results of transplanted embryo. The novel mutation of c. 156 G>T in CD40LG gene probably leads to XHIGM by nonsense-meditated mRNA decay (NMD), and complex PGT of preimplantation genetic testing for monogenic disease (PGT-M), aneuploidy (PGT-A), structural rearrangement (PGT-SR), and HLA-matching (PGT-HLA) can be performed in pedigree with both X-linked hyper IgM syndrome and Robertsonian translocation.

Noninvasive prenatal diagnosis by genome-wide haplotyping of cell-free plasma DNA

PurposeWhereas noninvasive prenatal screening for aneuploidies is widely implemented, there is an increasing need for universal approaches for noninvasive prenatal screening for monogenic diseases. Here, we present a cost-effective, generic cell-free fetal DNA (cffDNA) haplotyping approach to scan the fetal genome for the presence of inherited monogenic diseases. MethodsFamilies participating in the preimplantation genetic testing for monogenic disorders (PGT-M) program were recruited for this study. Two hundred fifty thousand single-nucleotide polymorphisms (SNPs) captured from maternal plasma DNA along with genomic DNA from family members were massively parallel sequenced. Parental genotypes were phased via an available genotype from a close relative, and the fetal genome-wide haplotype and copy number were determined using cffDNA haplotyping analysis based on estimation and segmentation of fetal allele presence in the maternal plasma. ResultsIn all families tested, mutational profiles from cffDNA haplotyping are consistent with embryo biopsy profiles. Genome-wide fetal haplotypes are on average 97% concordant with the newborn haplotypes and embryo haplotypes. ConclusionWe demonstrate that genome-wide targeted capture and sequencing of polymorphic SNPs from maternal plasma cell-free DNA (cfDNA) allows haplotyping and copy-number profiling of the fetal genome during pregnancy. The method enables the accurate reconstruction of the fetal haplotypes and can be easily implemented in clinical practice.

A systematic review on concurrent aneuploidy screening and preimplantation genetic testing for hereditary disorders: What is the prevalence of aneuploidy and is there a clinical effect from aneuploidy screening?

In assisted reproductive technology, aneuploidy is considered a primary cause of failed embryo implantation. This has led to the implementation of preimplantation genetic testing for aneuploidy in some clinics. The prevalence of aneuploidy and the use of aneuploidy screening during preimplantation genetic testing for inherited disorders has not previously been reviewed. Here, we systematically review the literature to investigate the prevalence of aneuploidy in blastocysts derived from patients carrying or affected by an inherited disorder, and whether screening for aneuploidy improves clinical outcomes. PubMed and Embase were searched for articles describing preimplantation genetic testing for monogenic disorders and/or structural rearrangements in combination with preimplantation genetic testing for aneuploidy. Original articles reporting aneuploidy rates at the blastocyst stage and/or clinical outcomes (positive human chorionic gonadotropin, gestational sacs/implantation rate, fetal heartbeat/clinical pregnancy, ongoing pregnancy, miscarriage, or live birth/delivery rate on a per transfer basis) were included. Case studies were excluded. Of the 26 identified studies, none were randomized controlled trials, three were historical cohort studies with a reference group not receiving aneuploidy screening, and the remaining were case series. In weighted analysis, 34.1% of 7749 blastocysts were aneuploid. Screening for aneuploidy reduced the proportion of embryos suitable for transfer, thereby increasing the risk of experiencing a cycle without transferable embryos. In pooled analysis the percentage of embryos suitable for transfer was reduced from 57.5% to 37.2% following screening for aneuploidy. Among historical cohort studies, one reported significantly improved pregnancy and birth rates but did not control for confounding, one did not report any statistically significant difference between groups, and one properly designed study concluded that preimplantation genetic testing for aneuploidy enhanced the chance of achieving a pregnancy while simultaneously reducing the chance of miscarriage following single embryo transfer. On average, aneuploidy is detected in 34% of embryos when performing a single blastocyst biopsy derived from patients carrying or affected by an inherited disorder. Accordingly, when screening for aneuploidy, the risk of experiencing a cycle with no transferable embryos increases. Current available data on the clinical effect of preimplantation genetic testing for aneuploidy performed concurrently with preimplantation genetic testing for inherited disorders are sparse, rendering the clinical effect from preimplantation genetic testing for aneuploidy difficult to access.

53. MEAN NUMBER OF ANALYZED EMBRYOS IN PGT-M CYCLES TO UNDERGO AT LEAST ONE EMBRYO TRANSFER

Introduction Preimplantation genetic testing for monogenic diseases (PGT-M) is a clinical method developed to prevent the transmission of monogenic inherited disorders to the future offspring. The reproductive risk of carriers of single gene disorders depends on the typology of the disorder, with the probability of affected conceptions ranging from 25% in recessive or X-linked diseases to 50% in dominant diseases. Therefore, the possibility of having embryos suitable for transfer and the consequent chances of ongoing pregnancy are strictly related to the number of embryos available for the genetic analysis. The aim of this study was to evaluate how many embryos need to be analyzed for having one or more genetically transferrable embryos. Material and Methods 86 carriers of genetic disorders performed 99 cycles of genetic analysis between January 2010 and September 2018. They were divided into two groups according to the typology of the disorder transmission. Group 1 included recessive and X-linked diseases (58 patients, 70 cycles of analysis), while with dominant disorders were in Group 2 (28 patients, 29 cycles of analysis). Maternal age was comparable in the two groups. Following blastocyst biopsy, embryos were vitrified to complete the genetic analysis. Results Clinical results were the following. For both groups, the mean number of analyzed embryos to have genetically transferrable embryos are reported in the following table: Conclusions Based on the reported data, we define for each case the minimum number of analyzable embryos needed to have one or more transferrable embryo depending on the typology of genetic disorder. Therefore, we eventually advise patients to undergo further oocyte retrievals for the best clinical outcome. Preimplantation genetic testing for monogenic diseases (PGT-M) is a clinical method developed to prevent the transmission of monogenic inherited disorders to the future offspring. The reproductive risk of carriers of single gene disorders depends on the typology of the disorder, with the probability of affected conceptions ranging from 25% in recessive or X-linked diseases to 50% in dominant diseases. Therefore, the possibility of having embryos suitable for transfer and the consequent chances of ongoing pregnancy are strictly related to the number of embryos available for the genetic analysis. The aim of this study was to evaluate how many embryos need to be analyzed for having one or more genetically transferrable embryos. 86 carriers of genetic disorders performed 99 cycles of genetic analysis between January 2010 and September 2018. They were divided into two groups according to the typology of the disorder transmission. Group 1 included recessive and X-linked diseases (58 patients, 70 cycles of analysis), while with dominant disorders were in Group 2 (28 patients, 29 cycles of analysis). Maternal age was comparable in the two groups. Following blastocyst biopsy, embryos were vitrified to complete the genetic analysis. Clinical results were the following. For both groups, the mean number of analyzed embryos to have genetically transferrable embryos are reported in the following table: Based on the reported data, we define for each case the minimum number of analyzable embryos needed to have one or more transferrable embryo depending on the typology of genetic disorder. Therefore, we eventually advise patients to undergo further oocyte retrievals for the best clinical outcome.

1. EVALUATION AND IMPLEMENTATION OF ONEPGT FOR MONOGENIC CONDITIONS

Introduction Preimplantation genetic testing for monogenic disorders (PGT-M) offers couples who carry a known inherited genetic condition the chance of having children free of the disorder. Currently, a bespoke genetic test is developed for each new condition which can take up to 6 months to implement; over 300 gene specific tests have been produced at our centre. OnePGT (Agilent) utilises genome-wide Next Generation Sequencing (NGS), allowing most genetic conditions to be tracked within families and embryos. Availability of a commercially developed genetic test eliminates the time involved with new test development. Evaluation of OnePGT for the testing of embryos for familial monogenic conditions has been undertaken. Material & methods Whole genome amplified (WGA) biopsy DNA was available for 33 embryos from 13 couples who previously underwent PGT-M; all embryo biopsies consisted of ∼5 trophectoderm cells. The genetic status of the embryos had been determined using an ISO15189 accredited STR haplotyping approach. 13 single gene disorders were tested including 9 autosomal dominant, 2 autosomal recessive and 2 X-linked. The embryo samples and respective reference family gDNA samples were processed using the OnePGT workflow, sequenced on a NextSeq550 and the data analysed using Alissa software for SNP haplotyping of the monogenic disorder, in conjunction with targeted assessment of copy number of the chromosome on which the gene was located, to aid interpretation of the monogenic results in embryos. Results OnePGT results were all in concordance with the outcomes from the STR results (33/33). The automated PGT-M call rate was 28/33 whilst 5 required manual inspection. Of these, 3 were because there was less than 1Mb between the genes to the end of the chromosome, 1 was due to the proximity of the gene to a chromosome crossover breakpoint and 1 was due to a reduced number of concordant SNPs which was caused by the low-level presence of both maternal haplotypes in the embryo. In one case, an affected embryo was successfully used as the reference for phasing for the couple and in another, the availability of chromosome copy number identified an embryo as having uniparental Inheritance. Conclusions The NGS approach of OnePGT has been successfully evaluated for determining the genetic status of embryos for monogenic disease. Due to the sequencing costs involved with NGS, it may not be merited when there is an established ‘in-house’ test. On balance, taking all laboratory aspects into consideration, OnePGT is a contender to evaluate new disorders. It is an anxious time for couples as they wait to know whether they have embryos suitable for replacement. Implementing OnePGT will eliminate test development and expedite genetic testing for couples requesting PGT-M for rare monogenic diseases. Declaration of Interest The OnePGT kits were provided by Agilent as part of an early access programme

52. MULTIPLE DISPLACEMENT AMPLIFICATION CAN INCREASE THE DIAGNOSTIC EFFICIENCY IN PGT-M FOR THALASSEMIA

Introduction Preimplantation genetic testing for monogenic diseases (PGT-M) has been widely conducted to prevent the birth of children genetically affected with thalassemia, especially in southeast China. However, due to the insufficient DNA template available from an embryo-level biopsy, its detection efficiency is still far from satisfactory. A suitable whole genome amplification (WGA) method is crucial to the success of PGT-M. This study aims to evaluate whether using multiple displacement amplification (MDA) as the first step can increase the diagnostic efficiency of PGT-M for thalassemia disorders. Material & methods This is a retrospective cohort study. All included patients underwent PGT-M cycles (n=939) for β-thalassemia-SEA or β- thalassemia in our center from January 2014 to February 2018. We divided the patients into two groups based on two different detection methods. For the polymerase chain reaction (PCR) group (n=428), after cell lysis we directly conducted fluorescent gap PCR for β-thalassemia-SEA diagnosis or multiplex nested PCR+ reverse dot blot (RDB) analysis for β- thalassemia diagnosis. For the MDA group (n=511), the whole genomes of single cells were directly amplified using MDA before fluorescent gap PCR or singleplex PCR+RDB procedures. Results A total of 7756 embryos were tested. The overall diagnostic efficiency of the MDA group was significantly higher than that of the PCR group (96.70% vs 86.60%, P Conclusions This study indicates that MDA, as the universal first step in PGT-M for β-thalassemia and β-thalassemia, can increase 10.10% diagnostic efficiency, thus may provide a better choice for the clinical PGT-M practice.

61. PREIMPLANTATION GENETIC TESTING FOR MONOGENIC DISEASE OF SPINAL MUSCULAR ATROPHY BY MULTIPLE DISPLACEMENT AMPLIFICATION: 11 UNAFFECTED LIVEBIRTHS

Introduction PGT-M has become an effective method for providing couples with the opportunity of a pregnancy with a baby free of spinal muscular atrophy (SMA). Multiple displacement amplification (MDA) overcome the innate dilemma of very limited genetic material available for PGT-M. This study mainly evaluated the use of MDA, combined with haplotype analysis and mutation amplification, in preimplantation genetic testing for monogenic disease (PGT-M) for families with SMA. Material and methods MDA was used to amplify the whole genome from single blastomeres or trophectoderm (TE) cells. Exon 7 of the survival motor neuron gene 1 (SMN1) and eleven STRs markers flanking the SMN1 gene were incorporated into singleplex polymerase chain reaction (PCR) assays on MDA products. Results Sixteen cycles (19 ovum pick-up cycles) of PGT-M were initiated in 13 patients. A total of 141 embryos were diagnosed: 90 embryos were biopsied at the cleavage stage and 51 embryos were biopsied at the blastocyst stage. MDA was successful on 94.44% (85/90) of the single blastomeres and on 92.16% (47/51) of the TE cells. And the PCR efficiency were 98.4%(561/570) and 100%(182/182), respectively. In addition, the average allele drop-out (ADO) rates were 13.3%(60/392) and 9.8% (11/112), respectively. The results for SMN1 exon 7 were all matched with haplotype analysis, which allowed an accurate diagnosis of 93.62% (132/141) embryos. Twelve families had unaffected embryos available for transfer and a total of 38 embryos were transferred in 20 embryo transfer cycles. Eight transfers were successful, resulting in an clinical pregnancy rate of 40%(8/20) and an implantation rate of 28.95%(11/38). Finally,11 healthy babies were born. Among them, 5 patients were singleton live births and and 3 patients had twin births. Conclusion Careful handling during the MDA procedure can improve subsequent PCR efficiency and reduce the ADO rate. We suggest that this protocol is reliable for increasing the accuracy of the PGT-M for SMA.

56. A NEW AGE IN PGT-M: A DECADE´S EXPERIENCE AND NEW CHALLENGES TO DEAL WITH

Introduction Genetic diagnosis has evolved dramatically mainly due to application of NGS approaches. Identification of mutations in different genes related with the disease and interpretation of pathogenicity of the new genetic variants found are some of the new challenges to face with. Preimplantation Genetic Testing for Monogenic Disorders (PGT-M) is applied for disease-causing genetic variants (Likely Pathogenic and Pathogenic). In parallel, Assisted Reproduction Techniques (ART) without PGT is not allowed in Spain if there is a high risk of transmitting a severe genetic disease. Therefore, clinical classification of genetic variant as pathogenic/bening is crucial for patients and it directly impacts in their future reproductive options. Spanish National legislation about ART/PGT-M approaches and best practice guidelines are currently not enough to solve some of these PGT-M requests. The aim of this work is to report our general experience since 2008 as a reference center for PGT-M in a Public Health System and the evolution of the complexity in the evaluation of the cases. Material&Methods From 2008-2017 a total of 372 cycles for PGT-M (202 families and 59 different monogenic diseases) including 5 PGT-HLA cases have been studied. Embryo biopsy was performed in 268 cases: 156 cases from 2007-2015 and 112 cases from 2016-2017. The genetic analysis was performed by cleavage aspiration and blastomere analysis was carried out by STR haplotype and Sanger sequencing. Clinical meeting minutes available from 2017 have been revised to analyze cases that were individually evaluated by a panel of specialists due to a Variant of Uncertain Significance (VUS) or diseases with an incomplete penetrance. Results During this decade the number of PGT-M requests has increased an average of 25% per year. Embryo genetic analysis was performed in 72% of the cycles with a 95% transfer rate. The Clinical Pregnancy rate (%per OR, % per Transfer) has been 23% and 29%, respectively. First cases requiring compound PGT-M of two different genetic alterations were requested between 2016-2017: 3 cases involved two different monogenic diseases and 1 case involved a monogenic disease and a chromosomal rearrangement (3,57% of the total PGT-M cases in this period). In 2017, four PGT-M cases with a VUS in COL1A2, MYH7, PKD1 and BRCA2 genes and two cases with an incomplete penetrance disease (Parkinson and FMF) needed previous evaluation by the specialist's panel. Based on the familial history, the VUS were reclassified as Probably Pathogenic in the four cases. The two cases with an incomplete penetrance were sent to the National Human Reproduction National Committee (CNRH) with a favorable resolution for the Parkinson´s case. Conclusions The PGT-M scenario is getting more complex especially due to that NGS-based analysis is becoming more informative and extensive. This complexity implies that additionally to the progress in technical approaches for PGT-M, the previous genetic/clinical evaluation of PGT families is of great importance. In this new age of PGT-M a multidisciplinary team of geneticists, gynecologists and occasionally other specialists, is becoming essential for an appropriate evaluation of PGT-M cases.

46. HAPLOTYPES BASED ON EIGHT VARIANTS IN SOUTHERN CHINA POPULATIONS WITH COMMON Α-THALASSEMIA DELETIONS AND THEIR APPLICATION TO PREIMPLANTATION GENETIC TESTING

Introduction Around 5 to 20% of the population in the provinces of southern China are carriers of β-thalassemia mutations. In our hospital, more than half of couples treated with preimplantation genetic testing for monogenic diseases (PGT-M) are β-thalassemia carriers. The most common mutations are deletion in β-thalassemia genes (HBA1 and HBA2), including –SEA, -α 3.7, and -α 4.2. Preimplantation genetic haplotyping (PGH) is a powerful technology for PGT-M that can detect allele drop-out (ADO) accurately by detecting numerous linked or unlinked polymorphic markers in DNA segments surrounding the genes of interest. However, there is a lack of normative data references in the selection of linkage sites for disease. Therefore, a mutation-specific haplotype analysis will improve the efficiency of PGT-M for β-thalassemia. Materials & methods A total of 113 couples with β-thalassemia deletion mutations were collected from the Reproductive Medicine Center in the First Affiliated Hospital of Sun Yat-sen University and the Reproductive and Genetic Hospital of CITIC-Xiangya. A pre-examination validation for PGT-M was performed on these. The SNPs with large difference between reference frequency and alternate frequency in 452 haplotypes with or without three kinds of deletions were filtered out. Candidate sites close to the β-thalassemia genes were selected, upstream and downstream of genes within 1 Mb. Then, the haplotypes model was established with several specific genotypes of SNPs inherited with the mutations. Subsequently, the model was verified to predict the genotypes in another 34 couples after PGT-M. In addition, the ADO ratio of each site in 255 embryos was analyzed. Results Eight candidate SNPs (rs2562164, rs2857997, rs2857998, rs116995933, rs149153770, rs3918352, rs1203977, and rs1203979) were identified for the haplotypes model constructed for different genotypes. SEA-I (GCGTAACA), α3.7-I (AGACGACA) and α4.2-I (AGACGGAT) haplotypes were detected in 92.2%, 50.9%, and 70.8% genotypes with corresponding mutations, respectively. On average, 42.7% sites were able to be used as key SNPs in the verification of another 34 PGT-M cohort haplotypes and genotypes. The SEA-I model was detected in 91.1% of genotypes with –SEA mutation. The α3.7-I model was concordant with 37.5% of genotypes with the -α3.7 mutation. The α4.2-I models were inherited with 87.5% of genotype with the -α4.2 mutation. The average ADO ratio of the eight SNPs in the 255 embryos was 2.3%. Conclusions The haplotype analysis for common β-thalassemia deletion mutations including the eight-site model improves the efficiency of PGT-M in the population of southern China. The establishment of standardized diagnostic procedures and systems can be further extended to other populations for PGT-M in the future.

59. PREIMPLANTATION GENETIC TESTING FOR HERITABLE CONNECTIVE TISSUE DISEASES

Introduction Heritable connective tissue disorders (HCTD) are a group of mostly autosomal dominant genetic diseases, characterized by cardiovascular involvement, tissue fragility, joint hypermobility and skin involvement. Some of them are associated with life threatening events such as aortic dissection and rupture, and a high mortality rate. We recently introduced Preimplantation Genetic Testing for monogenic disease (PGT-M) with a focus on HCTD and set up an explorative qualitative study to investigate the lived experiences and feelings of women, as well as their attitudes and psychological responses to PGT-M. Materials & Methods All couples are seen by a clinical geneticist and a psychologist, and blood samples of the couple are drawn for karyotyping, exclusion of carriership of cystic fibrosis, spinal muscular atrophy and fragile-X syndrome. Informative linked microsatellite markers flanking the causal (familial) mutation are identified for each couple. Once these parameters are defined, DNA, obtained from trophectoderm cells biopsied from a day 5-6 embryo, is genome-wide amplified. Subsequently, the informative linked microsatellite markers as well as the underlying disease causing mutation are analysed in each of these day 5-6 embryo's. In total, twelve HCTD couples have undergone the PGT-M procedure (FBN1: 4, SMAD3: 2, one each for TGFBR1, TGFB2, COL3A1, COL1A2, COL2A1 and WNT10A). In total, 52 embryos have been tested, 19 of them carried the healthy haplotype (microsatellite analysis) and did not carry the familial mutation (Sanger sequencing) and thus were suitable for transfer to the mother. Data of seven women, aged 26-39years, were collected by semi-structured interviews and interpreted by thematic analysis. At the time of the interviews, one participant had one child through PGT-M, two participants were pregnant after embryo transfer and four women did not have a successful embryo transfer leading to pregnancy yet. Results Our study shows that PGT-M is both physically and emotionally a demanding procedure. The main reason for choosing PGT-M is the severity of the disease. None of the couples wants to transmit the disease to their offspring. Since termination of pregnancy is not acceptable to them, PGT-M is preferred over prenatal diagnosis. The “drop-out race”, as all interviewed women call it, going from an encouraging number of available oocytes (after pick-up), to a limited number of useful embryos, is perceived as extremely stressful. Due to the frequent hospital visits and the prescribed strict timing of hormone injections, all women stated it is impossible to keep the PGT-M procedure hidden from their friends and colleagues. Women emphasize that receiving adequate information, during every step of the PGT-M process, is essential to learn more about the entire PGT-M trajectory. They also express the need of psychological support to help them in coping with this physical and psychological demanding technique. Conclusions PGT-M is a valuable option of reproductive technology for HCTD couples. Due to the significant emotional impact the PGT-M procedure causes, we advise that it should always go hand-in-hand with effective communication and psychological care in order to prevent distress in couples.

Transfer of embryos with positive results following preimplantation genetic testing for monogenic disorders (PGT-M): experience of two high-volume fertility clinics

To assess the experiences of two large fertility clinics in which embryos with positive results following preimplantation genetic testing for monogenic disorders (PGT-M) were transferred upon patient request, in order to explore the nature of the conditions for which these requests have been made and review ethical considerations. Retrospective review of previous embryo transfers at the NYU Langone Fertility Center and ORM Fertility was performed. Embryo transfers prior to May 2019 in which embryo biopsy and PGT-M occurred were reviewed, and transferred embryos that were positive for a monogenic disorder (excluding autosomal recessive carriers) were identified. Seventeen patients were identified who elected to transfer 23 embryos that tested positive for nine different monogenic disorders. Most of the embryos transferred were positive for disorders that are autosomal dominant (15/23), are adult-onset (14/23), are associated with reduced penetrance (16/23), and have available management to lessen symptom severity (22/23). Transfer of positive embryos most commonly occurred for hereditary cancer susceptibility syndromes (9/23 embryos), particularly hereditary breast and ovarian cancer syndrome. When unaffected embryos are not produced following in vitro fertilization with PGT-M, some patients request to transfer embryos with positive test results. The majority of transfers were for embryos positive for adult-onset, reduced penetrance diseases. As these requests will likely increase over time, it is essential to consider the practical and ethical implications.

Multi-centre evaluation of a comprehensive preimplantation genetic test through haplotyping-by-sequencing.

Can reduced representation genome sequencing offer an alternative to single nucleotide polymorphism (SNP) arrays as a generic and genome-wide approach for comprehensive preimplantation genetic testing for monogenic disorders (PGT-M), aneuploidy (PGT-A) and structural rearrangements (PGT-SR) in human embryo biopsy samples? Reduced representation genome sequencing, with OnePGT, offers a generic, next-generation sequencing-based approach for automated haplotyping and copy-number assessment, both combined or independently, in human single blastomere and trophectoderm samples. Genome-wide haplotyping strategies, such as karyomapping and haplarithmisis, have paved the way for comprehensive PGT, i.e. leveraging PGT-M, PGT-A and PGT-SR in a single workflow. These methods are based upon SNP array technology. This multi-centre verification study evaluated the concordance of PGT results for a total of 225 embryos, including 189 originally tested for a monogenic disorder and 36 tested for a translocation. Concordance for whole chromosome aneuploidies was also evaluated where whole genome copy-number reference data were available. Data analysts were kept blind to the results from the reference PGT method. Leftover blastomere/trophectoderm whole genome amplified (WGA) material was used, or secondary trophectoderm biopsies were WGA. A reduced representation library from WGA DNA together with bulk DNA from phasing references was processed across two study sites with the Agilent OnePGT solution. Libraries were sequenced on an Illumina NextSeq500 system, and data were analysed with Agilent Alissa OnePGT software. The embedded PGT-M pipeline utilises the principles of haplarithmisis to deduce haplotype inheritance whereas both the PGT-A and PGT-SR pipelines are based upon read-count analysis in order to evaluate embryonic ploidy. Concordance analysis was performed for both analysis strategies against the reference PGT method. PGT-M analysis was performed on 189 samples. For nine samples, the data quality was too poor to analyse further, and for 20 samples, no result could be obtained mainly due to biological limitations of the haplotyping approach, such as co-localisation of meiotic crossover events and nullisomy for the chromosome of interest. For the remaining 160 samples, 100% concordance was obtained between OnePGT and the reference PGT-M method. Equally for PGT-SR, 100% concordance for all 36 embryos tested was demonstrated. Moreover, with embryos originally analysed for PGT-M or PGT-SR for which genome-wide copy-number reference data were available, 100% concordance was shown for whole chromosome copy-number calls (PGT-A). Inherent to haplotyping methodologies, processing of additional family members is still required. Biological limitations caused inconclusive results in 10% of cases. Employment of OnePGT for PGT-M, PGT-SR, PGT-A or combined as comprehensive PGT offers a scalable platform, which is inherently generic and thereby, eliminates the need for family-specific design and optimisation. It can be considered as both an improvement and complement to the current methodologies for PGT. Agilent Technologies, the KU Leuven (C1/018 to J.R.V. and T.V.) and the Horizon 2020 WIDENLIFE (692065 to J.R.V. and T.V). H.M. is supported by the Research Foundation Flanders (FWO, 11A7119N). M.Z.E, J.R.V. and T.V. are co-inventors on patent applications: ZL910050-PCT/EP2011/060211- WO/2011/157846 'Methods for haplotyping single cells' and ZL913096-PCT/EP2014/068315 'Haplotyping and copy-number typing using polymorphic variant allelic frequencies'. T.V. and J.R.V. are co-inventors on patent application: ZL912076-PCT/EP2013/070858 'High-throughput genotyping by sequencing'. Haplarithmisis ('Haplotyping and copy-number typing using polymorphic variant allelic frequencies') has been licensed to Agilent Technologies. The following patents are pending for OnePGT: US2016275239, AU2014345516, CA2928013, CN105874081, EP3066213 and WO2015067796. OnePGT is a registered trademark. D.L., J.T. and R.L.R. report personal fees during the conduct of the study and outside the submitted work from Agilent Technologies. S.H. and K.O.F. report personal fees and other during the conduct of the study and outside the submitted work from Agilent Technologies. J.A. reports personal fees and other during the conduct of the study from Agilent Technologies and personal fees from Agilent Technologies and UZ Leuven outside the submitted work. B.D. reports grants from IWT/VLAIO, personal fees during the conduct of the study from Agilent Technologies and personal fees and other outside the submitted work from Agilent Technologies. In addition, B.D. has a patent 20160275239 - Genetic Analysis Method pending. The remaining authors have no conflicts of interest.

Robust Preimplantation Genetic Testing Strategy for Myotonic Dystrophy Type 1 by Bidirectional Triplet-Primed Polymerase Chain Reaction Combined With Multi-microsatellite Haplotyping Following Whole-Genome Amplification.

Myotonic dystrophy type 1 (DM1) is caused by expansion of the DMPK CTG trinucleotide repeat. Disease transmission to offspring can be avoided through prenatal diagnosis or preimplantation genetic testing for monogenic disorders (PGT-M). We describe a robust strategy for DM1 PGT-M that can be applied to virtually any at-risk couple. This strategy utilizes whole-genome amplification, followed by triplet-primed PCR (TP-PCR) detection of expanded DMPK alleles, in parallel with single-tube haplotype analysis of 12 closely linked and highly polymorphic microsatellite markers. Bidirectional TP-PCR and dodecaplex marker PCR assays were optimized and validated on whole-genome amplified single lymphoblasts isolated from DM1 reference cell lines, and tested on a simulated PGT-M case comprising a parent-offspring trio and three simulated embryos. Bidirectional DMPK TP-PCR reliably detects repeat expansions even in the presence of non-CTG interruptions at either end of the expanded allele. Misdiagnoses, diagnostic ambiguity, and couple-specific assay customization are further minimized by the use of multi-marker haplotyping, preventing the loss of potentially unaffected embryos for transfer.

Multiple displacement amplification as the first step can increase the diagnostic efficiency of preimplantation genetic testing for monogenic disease for β-thalassemia.

To evaluate whether using multiple displacement amplification (MDA) as the first step can increase the diagnostic efficiency of preimplantation genetic testing for monogenic disease (PGT-M) for β-thalassemia. This is a retrospective cohort study. All included patients underwent PGT-M cycles (n = 307) for β-thalassemia in our center from January 2014 to February 2018. We divided the patients into two groups based on two different detection methods. For the polymerase chain reaction (PCR) group (n = 115), multiplex nested PCR+ reverse dot blot analysis was performed directly after cell lysis. For the MDA group (n = 192), the whole genomes of single cells were directly amplified using MDA and then examined by singleplex PCR + reverse dot blot for β-thalassemia. A total of 2315 embryos were tested. The overall diagnostic efficiency of the MDA group was significantly higher than that of the PCR group (96.99% vs 88.15%, P < 0.001). The percentage of embryos available for transfer was significantly higher in the MDA group than in the PCR group (74.28% vs 64.98%, P < 0.001). Furthermore, the carrier embryo rate of the MDA group was significantly higher than that of the PCR group (50.11% vs 35.95%, P < 0.001). This study indicates that MDA, as the first step in PGT-M for β-thalassemia, can increase diagnostic efficiency.

Preimplantation genetic testing for more than one genetic condition: clinical and ethical considerations and dilemmas.

Which clinical and ethical aspects of preimplantation genetic testing for monogenic disorders or structural rearrangements (PGT-M, PGT-SR) should be considered when accepting requests and counselling couples for PGT when applied for more than one condition (combination-PGT; cPGT-M/SR)? cPGT is a feasible extension of the practice of PGT-M/SR that may require adapting the criteria many countries have in place with regard to indications-setting for PGT-M/SR, while leading to complex choices that require timely counselling and information. Although PGT-M/SR is usually performed to prevent transmission of one disorder, requests for PGT-M/SR for more than one condition (cPGT-M/SR) are becoming less exceptional. However, knowledge about implications for a responsible application of such treatments is lacking. Retrospective review of all (40) PGT-M/SR applications concerning more than one genetic condition over the period 1995-2018 in the files of the Dutch national PGT centre. This comprises all relevant national data since the start of PGT in the Netherlands. Data regarding cPGT-M/SR cases were collected by means of reviewing medical files of couples applying for cPGT-M/SR. Ethical challenges arising with cPGT-M/SR were explored against the background of PGT-M/SR regulations in several European countries, as well as of relevant ESHRE-guidance regarding both indications-setting and transfer-decisions. We report 40 couples applying for cPGT-M/SR of which 16 couples started their IVF treatment. Together they underwent 39 IVF cycles leading to the birth of five healthy children. Of the couples applying for cPGT, 45% differentiated between a primary and secondary condition in terms of perceived severity. In the light of an altered balance of benefits and drawbacks, we argue the 'high risk of a serious condition' standard that many countries uphold as governing indications-setting, should be lowered for secondary conditions in couples who already have an indication for PGT-M/SR. As a consequence of cPGT, professionals will more often be confronted with requests for transferring embryos known to be affected with a condition that they were tested for. In line with ESHRE guidance, such transfers may well be acceptable, on the condition of avoiding a high risk of a child with a seriously diminished quality of life. We are the first to give an overview of cPGT-M/SR treatments. Retrospective analysis was performed using national data, possibly not reflecting current trends worldwide. Our observations have led to recommendations for cPGT-M/SR that may add to centre policy making and to the formulation of professional guidelines. Given that the introduction of generic methods for genomic analysis in PGT will regularly yield incidental findings leading to transfer requests with these same challenges, the importance of our discussion exceeds the present discussion of cPGT. The research for this publication was funded by the Dutch Organization for Health Research and Development (ZonMw), project number: 141111002 (Long term safety, quality and ethics of Preimplantation Genetic Diagnosis). None of the authors has any competing interests to declare.