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To compare aneuploidy rates among in vitro fertilization (IVF) cycles using preimplantation genetic testing for monogenic disorders (PGT-M) and aneuploidy (PGT-A) compared with IVF cycles using PGT-A alone, and to determine the likelihood of obtaining at least one usable embryo in cycles using PGT-M+PGT-A compared with cycles using PGT-A alone. Retrospective cohort study. Single genetics laboratory. All IVF cycles for patients aged 18–45 undergoing PGT-A with or without concurrent PGT-M at a single genetics laboratory from November 2019 to March 2023. Use of PGT-M+PGT-A vs. use of PGT-A alone. Per cycle aneuploidy rate stratified by age, and per cycle likelihood of obtaining at least one usable embryo stratified by age and inheritance pattern of monogenic disease. A total of 72,522 IVF cycles were included; 4,255 cycles (5.9%) using PGT-M+PGT-A and 68,267 cycles (94.1%) using PGT-A alone. The PGT-M+PGT-A group was younger than the PGT-A alone group (<35 years old: 56.1% vs. 30.5%). The majority of PGT-M cycles were performed for autosomal dominant pathogenic variants (42.4%), followed by autosomal recessive (36.5%), X-linked dominant (13.3%), and X-linked recessive (7.5%). The median number of embryos biopsied was higher in PGT-A alone compared with PGT-M+PGT-A cycles for patients aged <35, but it was equivalent in all other age groups. Age stratified aneuploidy rates did not significantly differ between PGT-M+PGT-A compared with PGT-A alone cycles. The probability of having a usable embryo declined with increasing age across all inheritance patterns. Compared with PGT-A alone, PGT-M+PGT-A cycles for patients aged ≤40 across all inheritance patterns were significantly less likely to yield a usable embryo, except in cycles for autosomal recessive diseases in the 38–40 age group and X-linked recessive diseases in the 35–37 age group. There were no consistent differences seen between groups in patients over 40. Cycles for patients with autosomal dominant diseases had the lowest likelihood of yielding a usable embryo for patients aged <43. In vitro fertilization cycles using PGT-M+PGT-A have similar age-specific aneuploidy rates to those using PGT-A alone. Cycles for patients ≤40 using PGT-M+PGT-A are significantly less likely to yield a usable embryo compared with those using PGT-A alone. [ABSTRACT FROM AUTHOR]

Preimplantation genetic testing for monogenic diseases for adult-onset conditions is ethically permissible for various conditions, including when the condition is fully penetrant or confers disease predisposition. The Committee strongly recommends that a genetic counselor experienced with both preimplantation genetic testing for monogenic diseases and assisted reproductive technology therapies counsel patients considering such procedures. [ABSTRACT FROM AUTHOR]

The previous lack of national UK guidance on the use of Prenatal and Pre-implantation Genetic Testing (PND and PGT-M) for Monogenic Disorders for Germline Cancer Susceptibility Gene Variants (gCSGV) has led to disparities in care across the UK, and inequitable access to reproductive options for families living with cancer susceptibility syndromes. In 2023, the UK Cancer Genetics Group and Fetal Genomics Group of the British Society of Genetic Medicine developed joint consensus guidance seeking to provide healthcare professionals with a clear counselling framework to support individuals/couples during their reproductive decision-making process. The guidance is for healthcare professionals, individuals and couples with a gCSGV and their families, policy makers and charities supporting people with cancer susceptibility syndromes. Details about the consensus group participants, the main workshop's format, and the pre- and post-workshop nationwide surveys, are available in the full document. [ABSTRACT FROM AUTHOR]

Does inheritance of the fragile X mental retardation 1 (FMR1) premutation allele affect embryo morphokinetic development? A retrospective cohort analysis of 529 embryos from 126 IVF cycles of 39 FMR1 premutation female carriers undergoing preimplantation genetic testing for monogenic/single gene defects (PGT-M). Morphological and morphokinetic parameters obtained using a time-lapse monitoring system were compared between embryos that inherited the FMR1 premutation allele (FMR1 group, n = 271) and those who received the normal allele (normal group, n = 258). The following embryo outcome measures were compared: morphokinetic parameters up to day 3, start of blastulation time (tSB) for day 5 embryos and the rate of top-quality embryos on days 3 and 5. No differences were found in morphokinetic parameters between the groups from the time of intracytoplasmic sperm injection (ICSI) until a biopsy on day 3. The blastulation rate in the two groups was comparable. However, the start of blastulation was delayed in FMR1 embryos compared to that in the genetically normal embryos (median tSB: 104.2 h [99.3−110.3] versus 101.6 h [94.5−106.7], P = 0.01). In addition, the rate of top-quality FMR1 embryos was lower than that of genetically normal embryos (25.6% versus 38.8%, P = 0.04). Embryos that inherit the FMR1 premutation allele are of lower quality at the blastocyst stage compared with those that do not inherit the mutated allele. [ABSTRACT FROM AUTHOR]

Abstract Research question What is the incidence and origin of meiotic whole and segmental aneuploidies detected by karyomapping at a blastocyst stage in human-derived IVF embryos? What is the distribution of various types of errors, including rare chromosomal abnormalities? Design The incidence of chromosomal aneuploidies was assessed in 967 trophectoderm biopsies from 180 couples who underwent 215 cycles of IVF with preimplantation genetic testing for monogenetic disease with a known causal mutation with a mean maternal age of 32.7 years. DNA from both parents and a reference sample was genotyped together with the analysed trophectoderm samples by karyomapping (single-nucleotide-polymorphism-based array). Results Chromosomal abnormalities were detected in 31% of the analysed samples. At least one whole chromosomal aneuploidy was detected in 27.1% of the trophectoderm biopsies, whereas a segmental aneuploidy was detected in 5.1% of the trophectoderm biopsies. Our results reveal that segmental aneuploidies predominantly affect paternally derived chromosomes (70.4%; P < 0.01) compared with whole chromosomal aneuploidies that more frequently affect maternally derived chromosomes (90.1%; P < 0.0001). Also, the frequency of meiosis I (MI) and meiosis II (MII) errors was established in meiotic trisomies; MI errors were observed to be more frequent (n = 102/147 [69.4%]) than MII errors (n = 45/147 [30.6%]). Conclusions Karyomapping is a robust method that is suitable for preimplantation genetic testing for monogenetic disease and for detecting meiotic aneuploidies, including meiotic segmental aneuploidies, and provides complex information about their parental origin. Our results revealed that segmental aneuploidy more frequently affects paternal chromosomes compared with whole chromosomal aneuploidy in human IVF embryos at the blastocyst stage. [ABSTRACT FROM AUTHOR]

Preimplantation genetic testing of monogenic disease (PGT-M) is technically challenging, because extremely small amount of biomaterial increases allele dropout (ADO) rates, contamination significance, failed amplification (FA) events. We report here our experience of PGT-M in "Genetico" center in Russia. A retrospective analysis of all requests and cycles of PGT-M referred to our center was performed. Embryo biopsies from 29 medical centers were sent for PGT-M. Embryos were mostly biopsied on day 5-6. PGT-M assays combine direct diagnosis of the pathogenic variants and linkage analysis of highly heterozygous STRs. The nested PCR was used for DNA amplification from different sources: single cells or whole genome amplification (WGA) products of embryo biopsy, total DNA. PGT-A by NGS or aCGH was performed for unaffected embryos upon patient's request. Of 109 couples referred to our center for PGT-M, 92 completed preliminary test for PGT-M for 42 genetic condition and HLA gaplotyping: 24 autosomal-dominant (AD) requests, 54 autosomal recessive (AR), 13 X-linked requests. The most frequent indication was spinal muscular atrophy (17 couples) and cystic fibrosis (8 couples). All these couples referred for PGT-M not after preconception screening, but because of affected child birth. We performed 85 PGT-M cycles with 413 embryos. Almost all of these samples undergo WGA for possibility to combine PGT-M with PGT-A, also each test-system was validated for single-cell either. The WGA failed in 16 cases (3,9%). The reliability was decreased for 12 embryo results (3%), because of increased number of ADO or marker FA events. Median number of markers included in test-systems was 12 and for embryo analysis it was 10. These highly informative test-systems contributed to low number of inconclusive results – only for 7 samples (1,8%) (Girardet et al. 2018). The recombination events are not the reason to decrease the reliability if sufficient number of informative markers employed. The recombination event was revealed in 39 samples (9,4%). Median distance between markers of test-system (2,97 Mb) suggests lower recombination rate 3,75%. Of all analyzed embryos 131 (31,7%) did not inherited any of pathogenic alleles (AD – 61; AR – 48; X-linked – 11), 135 (49%) were carriers (AR – 122; X-linked – 11), 136 (32,9%) were affected (AD – 49; AR – 67; X-linked – 10). For 156 (58,6%) unaffected embryos PGT-A was performed and 91 (34,1%) were suitable for transfer. Chromosomal abnormalities for three embryos were revealed at PGT-M stage and confirmed by PGT-A. At the moment we have information about 43 transfers, 19 pregnancies and 7 healthy births and no affected pregnancy or birth. Highly informative test system and accurate analysis of results can lead to both - high accuracy of obtained results and decreased number of embryos, that were rejected because of inconclusive results. PGT-M appears to be more robust analysis than PGT-A. [ABSTRACT FROM AUTHOR]

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. 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. 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. 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. [ABSTRACT FROM AUTHOR]

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. 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. 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. 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. [ABSTRACT FROM AUTHOR]

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. 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. 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. 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. The OnePGT kits were provided by Agilent as part of an early access programme [ABSTRACT FROM AUTHOR]

Couples requesting PGT-M involving a de novo mutation pose a challenge when the PGT-M test used incorporates linkage analysis, as parental samples cannot be used to determine the mutant and normal allele haplotypes. In our experience, de novo mutations represent approximately 6-7% of clinical referrals, and thus it is important to have strategies in place to be able to offer PGT-M for these patients. Historically, we have employed two main strategies for these couples. If the mutation in question was paternal, we would perform single sperm analysis to determine the mutant haplotype. When the mutation was maternal, we would perform single chromosome sorting to determine the mutant haplotype. We would then confirm the phase when we obtained samples from clinical embryos. More recently, we began to question if determining phase prior to a PGT-M cycle was necessary, as we were confirming the phase as part of the cycle. We decided to evaluate if it was quicker and simpler to aim to determine phase directly from the embryos created as part of the clinical PGT-M. Haplotype determination was performed for the cases, utilising biopsies from clinically usable embryos. In addition, in order to obtain enough sample numbers to determine phase, when there were less than 4-5 embryos biopsied, we also requested samples from embryos with poor morphological development. Karyomapping was the PGT-M test performed, in combination with a method to detect the mutation from MDA amplifications from the biopsied samples. A grandparental DNA reference was available in all cases to evaluate SNP coverage prior. We obtained 3-7 samples from each of 14 cases to determine mutant and normal haplotypes. On 8/14 (57%) occasions, there were not enough samples from clinically usable embryos to provide us with the required sample numbers, so additional samples from embryos with poor morphological development were requested. Phase was established in all 14 cases. In all 12/14 (86%) cases the de novo mutation had a grandpaternal origin consistent with previous cases showing 89% of these de novo mutations have arisen in a sperm. This strategy proved successful in all 14 cases, allowing PGT-M to proceed without any delay. Additional samples from morphologically poor embryos were required 57% of the time, in order to obtain the necessary sample numbers to determine the mutant haplotype. This strategy can be employed to facilitate fast access to Karyomapping based PGT-M for cases involving de novo mutations, with a back-up strategy of using the above mentioned other methods if sample numbers are lacking. [ABSTRACT FROM AUTHOR]

PGT-M may presently be applicable for any inherited disorder for which sequence information or relevant haplotypes are available for the detection by direct mutation analysis or haplotyping in oocytes or embryos. However, these approaches cannot be applied in cases of de novo mutations (DNM) in parent(s) or affected children, as neither origin nor relevant haplotypes are available for tracing the inheritance of this DNM in single cells biopsied from embryos or in oocytes. On the other hand, with the improved awareness of PGT, an increasing number of couples request PGT, without any family history of the genetic disease that has been first diagnosed in one of the parents or in their affected children. So special PGT strategies are required for the genetic conditions determined by DNM. We developed PGT strategies for DNM which were applied for 277 families with 83 different genetic conditions. The majority, 256 of them, were determined by dominant mutations, with only 4 by autosomal recessive and 21 - by X-linked DNM. It is of interest, that despite the expected predominance of dominant DNM of a paternal origin, with the increasing proportion of older paternal partners in the modern society, there was a comparable proportion of DNM of the paternal (138 couples) and maternal origin (110 couples). The latter presents a particular challenge in developing a PGT strategy, frequently requiring polar body testing to determine maternal haplotypes. The other challenge is presented by gonadal mosaicism detected in increasing number of PGT parents. In addition, up to 10% of the tested DNM (29 patients) were first detected in the affected children, with no evidence of detectable mutation in parents, despite the finding the corresponding mutant haplotype associated with normal allele. PGT strategies for these families were different depending on the origin of DNM, and included an extensive DNA analysis of the parents and affected children prior to PGT, with the mutation verification, polymorphic marker evaluation, whole- and single-sperm testing, and PB analysis in order to establish the normal and mutant haplotypes, without which PGT cannot be performed. In cases of DNM of paternal origin, the DNM was first confirmed on the paternal DNA from blood and total sperm, followed by single-sperm typing to determine the proportion of sperm with DNM and relevant normal and mutant haplotypes. For a higher reliability of testing, the relevant maternal linked markers were also detected, to be able to trace for possible shared maternal and paternal markers. In cases of DNM of maternal origin, DNM was first confirmed in maternal blood, and PGT was performed, when possible, by PB analysis, to identify the normal and mutant maternal haplotypes. Also, in order to trace the relevant paternal haplotypes, single-sperm typing was performed, whenever possible, for avoiding misdiagnosis caused by possible shared paternal and maternal markers. In cases of DNM-detected first in children, the mutation was verified in their whole blood DNA, followed by testing for the mutation in paternal DNA from blood, total and single sperm. So, in contrast to previous PGT practice, performing PGT for DNM required extensive preparatory DNA work before performing the actual PGT, with the additional tests including single-sperm analysis and the requirement of performing sequential PB1 and PB2, followed by blastocyst analysis. Overall, we performed 516 PGT cycles for DNM for 277 couples, which resulted in pre-selection and transfer of 678 DNM-free embryos in 464 cycles (average of 1.4 embryos per transfer) yielding 262 (56%) unaffected pregnancies, with only 25 (9.5%) spontaneous abortions, and birth of 265 healthy children, confirmed to be free of DNM tested This is the world's largest series of PGT for DNM, which could not be performed by traditional approaches, due to unavailability of family history and lack of any affected family member to identify the origin of mutation and trace the inheritance of the mutant and normal alleles in oocytes and embryos. However, as demonstrated the specific strategies may be developed in search for the possible origin of DNM and relevant haplotypes as the basis for developing a PGT design for each particular couple with DNM, allowing a highly accurate pre-selection of oocytes and embryos free from DNM. [ABSTRACT FROM AUTHOR]

In Japan, preimplantation genetic testing for monogenic (PGT-M) can only be done under Japan Society of Obstetrics and Gynecology (JSOG) approval facility. Inside the approval facility, case has to get an approval from JSOG. Due to such strict condition, there are only 5 facilities that have experienced the PGT-M. Even to the genetic diseases that are subjects of PGT-M among other countries, it is not so in Japan and only limited to severe genetic diseases with onset at childhood. In this study, we will consider the issues of PGT-M in Japan from our genetic counseling and their subsequent decision by couples who wanted PGT-M at our clinic. The subject is 31 couples who made reservation of consultation with our clinic for the purpose of PGT-M from August 2014. In order to obtain approval of PGT-M by JSOG, the couple need to receive genetic counseling not only at the PGT-M implementation facility but also at two different facilities. 21 couples out of 30 couples who received genetic counseling at our clinic wished to apply PGT-M to JSOG. A couple with a child with an Alpha-thalassemia X-linked mental retardation syndrome failed to undergo consultation for genetic counseling at our clinic because the condition of the child worsened. After genetic counseling at our clinic, seven couples did not want PGT-M. One couple with a child with congenital glycosylation disorder of glycosylation (type Ik) gave up PGT-M because it was difficult to attend our clinic with treatment of the affected child since it took more than 3 hours to our clinic. Four couples abandoned PGT-M because it takes a long time to approve. Also, in a couple with Myotonic dystrophy 1 male patient, the fact that they are not suited for PGT-M was not easily accepted and they had to be re-consulted by other facilities before they could accept the fact. The couple of congenital myopathy central core disease was selected for PGT-M because of adult onset disease. In other two couples, one couple applied for PGT-M at different facility, and the other has not yet been able to make the decision after genetic counseling. Although there may be couples who want to take PGT-M, there are not enough facilities that have experienced the PGT-M in Japan. In addition, indication of PGT-M in Japan is severely limited to diseases in which symptoms that strongly disdain daily life are developed and the survival is dangerous before reaching adults. In the future, based on overseas adaptation criteria, Japan's adaptation criteria should be relaxed [ABSTRACT FROM AUTHOR]

Genome-wide haplotyping strategies, such as karyomapping, haplarithmisis and OnePGT, have paved the way for comprehensive PGT, leveraging PGT-M, PGT-A and PGT-SR in a single workflow. Nevertheless, family-specific challenges can complicate the implementation of current methods. First, to enable haplotype reconstruction, genotyping the prospective parents and direct family members, i.e. offspring of the couple or their parent(s), is required. However, if these references are not available or suitable for phasing, such approaches cannot be used, prompting investigation towards alternative phasing modules. Additionally, the advancement of current technologies leads to the identification of an increasing number of variants of unknown significance. Development of novel analysis approaches can assist further evaluation and interpretation of such variants, based on the familial segregation of the variant, in order to assess if PGT is an appropriate clinical option. Twelve couples i) burdened with a known hereditary genetic disorder participating in the genome-wide haplotyping-based PGT-M program and ii) with at least one parental sibling from the parental side carrying the genetic disorder available, were recruited for the development of the new method. Genotyping data from both prospective parents and parental sibling(s) were used to trace the shared and/or different allele and impute the disease-carrying allele based on identity-by-state (IBS) information. Concurrent haplotyping and copy number typing of embryo biopsies was then performed using an adapted version of siCHILD/haplarithmisis. Six out of 12 (50%) PGT-M couples could be phased using parental sibling(s). This is in line with the theoretical expectations of allele-sharing between sibling-pairs. Genome-wide haplotypes and copy-number profiles generated for each embryo using the new phasing approach were consequently compared to the clinical results, showing 100% concordancy. Clinical implementation of the method has resulted in the analysis of 7 families so far. Additionally, IBS analysis has enabled the inclusion of two special cases in the PGT program. In the first, PGT-M was offered for an X-linked disorder. The female partner had karyotype 47,XXX with presence of both maternal X chromosomes, so that no distinction could be made between the affected and unaffected allele. In the second, IBS analysis revealed the de novo and consequently pathogenic nature of an autosomal dominant variant in the maternal grandmother, allowing PGT. IBS analysis in the context of PGT can be proven to be advantageous on several levels. First, it allows offering genome-wide linkage analysis-based comprehensive PGT to families lacking standard phasing reference(s). A rapid pre-PGT work-up can define whether the couple can benefit from this technology. Importantly, by including more than one extended family member, the chance of obtaining informative results in the interrogated locus increases. Furthermore, by following the familial segregation of the variant of interest using IBS information, more evidence can be obtained regarding its pathogenicity. This is important for making the decision if the specific indication is valid for PGT. [ABSTRACT FROM AUTHOR]

Recently, we have developed PGD-Seq, which is a solution for PGT-M and PGT-SR analysis, which can be coupled to PGT-A. PGD-Seq has several advantages over other solutions: (1) PGD-Seq is designed to work widespread in any family from any ethnicity, so no specific customization is needed; (2) the same solution can be used to detect embryos with balanced translocations; (3) it is possible to detect even small unbalanced translocations, increasing the resolution of standard PGT-SR; (4) it can be combined with PGT-A in the same run and (5) it is an easy workflow with possibilty of automation. Here we present data about the performance of PGD-Seq in challenging situations. Close to 200 cases of PGT-M and PGT-SR have been analyzed with PGD-Seq solution. Each PGD-Seq panel has been designed for a specific gene or region with a complex algorithm in order to select informative SNPs among different ethnicities. Finally, each panel involves around 200 pre-selected SNPs. Informativity study and embryo analysis have been done by PGD-Seq simultaneously in some cases. Frequently, PGD-Seq was combined with PGT-A. Up to know, more than 150 gene analysis and 200 case studies have been developed with PGD-Seq. Among different cases, some of them were very challenging. Firstly, the same panel was used in different ethnicities. Moreover, it was used in some couples with high level of consanguinity, where we were even able to identify informative SNPs. Due to the high density of SNPs in the regions studied, we were able to identify recombinant embryos in few different cases. Finally, due to the ability of combining direct and indirect testing, PGD-Seq was used in cases without any family member available in order to complete the informativity test, or with complex family combinations. Regarding to PGT-SR, the solution was able to differentiate normal from balanced embryos in several cases. Nowadays, FISH is commonly used in several labs for specific case studies, especially in those with a small portion of the chromosome affected. However, just with PGD-Seq, we were able to identify those small aberrations. Finally, the solution was used even in a case with an inversion. The used of PGD-Seq shows its robustness in very challenging situations, without any previous setup. Additionally, the target approach instead of whole genome one keeps the costs of the test low enough to make it affordable for most of the couples. [ABSTRACT FROM AUTHOR]

Couple who are both β-thalassemia carriers usually come to PGT-M test for healthy offsprings. STR-linkage analysis is a well-known solution but Allele Drop-Out, high mutation rate and stutter peaks are still major challenges. Karyomapping is a common technique but it's costly and not be combined with PGT-A test, which may result in multiple biopsy or unaffected but aneuploidy embryos. In this study, we report two cases of β-thalassemia PGT test which uses single nucleotide polymorphisms (SNPs) for linkage analysis. By using specific primers in WGA process, the Allele Drop-out rate decreases to < 1%. This strategy is enable to combine PGT-M and PGT-A in one single test which allows to select ploidy and unaffacted embryos for transplant. Two β-thalassemia-carrier couples underwent IVF procedure, resulted in 11 embryos. Specific primers were desinged to amplify whole HBB-coding region and SNPs within 300kb flanking the HBB gene. Day 5 biopsied samples were used for whole genome amplification with designed specific primers in order to decrease ADO in HBB gene region and SNP markers, using DOPlify kit (RHS). WGA product and specific primers were used for PCR reaction to enrich HBB region and interested SNPs. Mix of WGA and enrichment PCR product was used for libraries preparation using Nextera XT DNA Library Prep Kit (Illumina) and sequenced on Miseq System. Nexus copy number was used for CNV analysis andMiseq Reporter was used for mutation detection and SNP calling. For the total of 11 samples, three samples resulted in aneuploidy. HBB mutation detection and SNP calling results showed no ADO in all 11 samples, with 3 affected embryos and 8 unaffected embryos. We have successfully designed a procedure which allows to combine PGT-A and PGT-M in one single test. This test is more cost-effective and decreases turn-around time compared with other techniques. ADO rate of HBB region and SNP makers is approximately 0%, which make it easier and more reliablefor ADO confirmation among embryos in the same cycle. [ABSTRACT FROM AUTHOR]

It is a common practice to perform PGT-M for one or two disorders at the same test, but it is the first PGT performed simultaneously for 5 different conditions, which is presented below. Consanguineous couple presented for PGT-M to avoid the risk of producing another affected child homozygous for four different autosomal recessive conditions identified in their previous offspring, including: (1) early infantile epileptic encephalopathy 5 (EIEE5), caused by SPTAN1 mutation; (2) xeroderma pigmentosum-complementation group C (XPG), caused by ERCC5 mutation; (3) congenital merosin–deficient muscular dystrophy 1A (MDC1A), caused by LAMA2 mutation; and (4) phenylketonuria (PKU) caused by PAH mutation. As parents requested also aneuploiidy testing, PGT design was to combine PGT-A and PGT-M, involving mutation and linked marker testing by multiplex nested PCR, to avoid the undetected ADO of each of the genes tested. Overall, 12 of 16 embryos reaching the blatocyst stage were tested together with NGS for PGT-A, of which 10 were affected, including 6 affected by one mutation, and four by two mutations. Only 2 embryos were unaffected carriers of all 4 gene mutations, of which one was with trisomy 13, so only a single embryos euploid and carrier of all 4 gene mutations was transferred, resulting, resulting in birth of a healthy unaffected baby. Although a cumulative risk of couple for producing an offspring affected by all the four conditions is only 0.4%, the couple still has been very unfortunate to produce such a child in their natural cycle. Although the chance for detecting unaffected embryo was also not high enough (31.6%), especially by added 50% risk for aneuploidy (15.8%), still one of 12 embryos tested was both euploid and normal carrier of all the mutations, resulting in a healthy, unaffected child. This is the world's first PGT for 5 different genetic conditions (EIEE5, XPG, MDC1A, PKU and aneuploidy) in a single test, resulting in a transfer of euploid embryo free of all the conditions tested, demonstrating feasibility and accuracy of simultaneous combined PGT for multiple genetic conditions. [ABSTRACT FROM AUTHOR]

A large number of pathogenic mitochondrial DNA (mtDNA) mutations have been identified and are implicated in a variety of disorders. As mtDNA is maternally inherited, women with pathogenic mtDNA mutations are likely to have affected children, the severity of the phenotype depending on the heteroplasmy proportion. A systematic meta-analysis showed that there is a ≥ 95% chance of being unaffected at a mutant level of ≤ 18% (Hellebrekers et al, 2012). PGT-M can be used to identify embryos with mutation loads below this phenotypic threshold. The PG-Seq™ kit yields superior coverage of mtDNA and may allow combined PGT-A and PGT-M for mtDNA diseases from single biopsies. Two cell lines, a reference and "mutant", with mitochondrial single nucleotide variants (SNV) were selected. Six 5-cell samples from each cell line were whole genome amplified (WGA). The replicates were pooled before the cell lines were mixed in proportions ranging from 0% to 100% (mutant), in increments of 10%. Libraries were prepared for each of the pools prior to sequencing using the MiSeq instrument (Illumina). SNV sites with < 10x depth across all samples were removed, leaving 31 SNV sites. The proportion of mutant SNV at each site was calculated and analysed. The data were corrected for variance introduced by pipetting error in pooling. These corrections were only applied to samples with proportions of 40%, 50%, and 60%, and subsequent analyses were restricted to these data. To model data at different read depths we combined data across sites. The average (± sd) mitochondrial read count for cell line samples was 5037 (± 1083). After filtering, the average (± sd) depth of coverage for the cell line samples at the SNV sites was 25.8x (± 12.5). Combining SNV data in silico to achieve a read depth typical of a standard PG-Seq™ kit 48 sample run using a single embryo biopsy (500,000 reads total), the observed SNV proportion was within 25% of the expected proportion in all cases. Combining SNV data to achieve a read depth of 5x a standard PG-Seq™ kit 48 sample run, the observed SNV proportion was within 11% of the expected proportion in all cases. This means that in any case where a heteroplasmy is detected at 7% or lower, including no detection, the mutant load should be below the phenotype threshold. The superior amplification of mtDNA achieved by the PG-Seq™ kit may allow combined PGT-A and PGT-M for mtDNA analysis. Combining PGT-A and PGT-M in this novel way would provide a streamlined workflow over currently available workflows. While accuracy is dependent on read depth, read depth can be increased by modulating sequencing throughput or by alternate means such as using PerkinElmer's Target Sequence Enrichment protocol. Due to variable but consistent depth of coverage across the mtDNA, some sites will be more suited for analysis by this method than others. [ABSTRACT FROM AUTHOR]