Results Of Preimplantation Genetic Testing Research Articles

Comprehensive preimplantation genetic testing for balanced insertional translocation carriers

BackgroundBalanced insertional translocations (BITs) can increase the risk of infertility, recurrent miscarriages or neonatal birth defects due to chromosomal imbalances in gametes. However, studies on preimplantation genetic testing (PGT) for...

SHaploseek is a sequencing-only, high-resolution method for comprehensive preimplantation genetic testing

Recent advances in genomic technologies expand the scope and efficiency of preimplantation genetic testing (PGT). We previously developed Haploseek, a clinically-validated, variant-agnostic comprehensive PGT solution. Haploseek is based on microarray genotyping of the embryo’s parents and relatives, combined with low-pass sequencing of the embryos. Here, to increase throughput and versatility, we aimed to develop a sequencing-only implementation of Haploseek. Accordingly, we developed SHaploseek, a universal PGT method to determine genome-wide haplotypes of each embryo based on low-pass (≤ 5x) sequencing of the parents and relative(s) along with ultra-low-pass (0.2–0.4x) sequencing of the embryos. We used SHaploseek to analyze five single lymphoblast cells and 31 embryos. We validated the genome-wide haplotype predictions against either bulk DNA, Haploseek, or, at focal genomic sites, PCR-based PGT results. SHaploseek achieved > 99% concordance with bulk DNA in two families from which single cells were derived from grown-up children. In embryos from 12 PGT families, all of SHaploseek’s focal site haplotype predictions were concordant with clinical PCR-based PGT results. Genome-wide, there was > 99% median concordance between Haploseek and SHaploseek’s haplotype predictions. Concordance remained high at all assayed sequencing depths ≥ 2x, as well as with only 1ng of parental DNA input. In subtelomeric regions, significantly more haplotype predictions were high-confidence in SHaploseek compared to Haploseek. In summary, SHaploseek constitutes a single-platform, accurate, and cost-effective comprehensive PGT solution.

A novel embryo biopsy morphological analysis and genetic integrality criterion system significantly improves the outcome of preimplantation genetic testing.

Whileefforts have been made to establish blastocyst grading systems in the past decades, little research has examined the quality of biopsy specimens. This study is the first to correlate the morphology of biopsied trophectoderm (TE) cells to their quality and subsequent genetic testing results of preimplantation genetic testing (PGT), through an innovative Morphological Analysis and Genetic Integrality Criterion (MAGIC) system. Biopsied TE cells were first evaluated according to the MAGIC procedure, followed by whole-genome amplification (WGA) and library construction, and then sequenced using the Illumina X Ten Platform. Copy number variation (CNV) and allele drop-out (ADO) rates as well as test failure rates were compared and analyzed. Our data explores the relationship between TE cell morphology and its quality and final genetic testing outcome, which is established based on the MAGIC system. MAGIC guarantees that only high- or good-quality TE cells are used for genetic testing to generate excellent data uniformity and lower ADO rates. Low-quality cells containing biopsied TE cell mass are responsible for the "background noise" of CNV analysis. The MAGIC application has effectively decreased the false-positive mosaicism, hence to ensure the stability and veracity of detection results, to avoid misdiagnoses, and to improve accuracy, as well as to avoid re-biopsy procedures. The study also contributes to understand how the IVF laboratory and the molecular biology laboratory depend on each other to achieve good-quality PGT results, which are clinically relevant for the patients.

P-132 Does the automated blastocyst assessment system, iDAScore®, provide valuable data for predicting the results of preimplantation genetic testing for aneuploidy (PGT-A)?

Abstract Study question What iDAScore® could indicate the presence of euploidy in PGT-A? Is it a valid predictor of the result of PGT-A? Summary answer PGT-A results with an iDAScore® of 8.3 or higher can indicate euploidy. However, patient age and trophectoderm (TE) grade can be effective predictors as well. What is known already PGT-A was approved as a general test in Japan in 2022. Currently, only patients who meet the criteria of Japan Society of Obstetrics and Gynecology can undergo the test. It is expected that the test will improve the live birth rate and reduce the miscarriage rate. The iDAScore® uses artificial intelligence and deep learning to score blastocysts. It is anticipated to be more objective than is morphological evaluation, which varies among individuals. Unlike PGT-A, the iDAScore® can assess embryos without cellular invasion. However, only a few reports have investigated the association between PGT-A and iDAScore® results. Study design, size, duration The study was performed retrospectively on 3,781 cycles of in vitro fertilization (IVF) conducted at our clinic between August 2020 and October 2022. Of the total of 5,684 embryos derived from conventional IVF or intracytoplasmic sperm injection that were cultured in a time lapse incubator, 293 blastocysts were used in the study. Participants/materials, setting, methods On the basis of their PGT-A results, the blastocysts were divided into euploidy and aneuploidy groups. The mean age of patients in each group, the methods of insemination, and the median iDAScore® were evaluated. The area under the curve (AUC) and the cut-off value were calculated from the PGT-A and iDAScore® results. A logistic regression analysis of the predictive parameters of PGT-A results, including the iDAScore®, was also performed. Main results and the role of chance Patients’ mean ages in the euploidy and aneuploidy groups were 38.5 and 41.5 years, respectively. This was significantly higher in the aneuploidy group (P<0.001). There was no significant difference in the stage of maturity at oocyte retrieval (P = 0.302). Similarly, no significant differences were associated with method of insemination (P = 0.961). The median iDAScore® for the euploidy group was 8.7; for the aneuploidy group, it was 7.8. This result was significantly higher in the euploidy group (P<0.001). The AUC, calculated from PGT-A results and the iDAScore® with receiver operating characteristic curves, was 0.685 (95% CI [0.617−0.754]). The cut-off value was 8.3. The logistic regression analysis of age, stage of maturity, insemination method, timing of blastocyst freezing, diameter before blastocyst freezing, inner cell mass, TE grade in the Gardner classification, and iDAScore® as parameters for the PGT-A results indicated significant differences for age and TE grade (age: P<0.001, odds ratio [95%CI]: 1.320 [1.19-1.45]; TE grade 2: P = 0.03, odds ratio [95%CI]: 2.480 [1.11-5.57]; TE grade 3: P = 0.02, odds ratio [95%CI]: 3.740 [1.27-11.00]), while no significant differences were associated with the iDAScore®. Limitations, reasons for caution The iDAScore® is likely to depend on the timing of blastocyst freezing. Furthermore, the iDAScore® utilised in this study was the score of blastocysts that fulfilled our freezing criteria; the blastocyst that reaches a size of at least 150-160 µm in diameter on Day 5,6, or 7 is frozen. Wider implications of the findings An iDAScore® of 8.3 or above could indicate a euploidy PGT-A result. However, the logistic regression analysis indicated that age and TE grade were more significant predictors than the iDAScore® was. Therefore, it is difficult to predict PGT-A results from the iDAScore® itself. Trial registration number Not applicable

Cell-free deoxyribonucleic acid analysis in preimplantation genetic testing

Detection of chromosomal aneuploidies and monogenic disorders in preimplantation embryos is essential for selecting the best embryo for transfer during invitro fertilization to achieve a healthy pregnancy. Preimplantation genetic testing (PGT) is typically performed on preimplantation embryos to select a genetically normal embryo for transfer. A trophectoderm biopsy is necessary for PGT; this is an invasive procedure to the embryo that requires specialized equipment and highly trained embryologists, resulting in high costs associated with invitro fertilization treatment. Moreover, the biopsy procedure may increase the likelihood of developing pregnancy complications, such as preeclampsia and hypertensive disorders. Therefore, there is a need for noninvasive embryo screening strategies. The presence of cell-free deoxyribonucleic acid in the embryo culture medium presents an opportunity to screen for genetic abnormalities. Cell-free deoxyribonucleic acid is released by embryos in the latter stages of preimplantation development, and its analysis has been proposed as a noninvasive approach for PGT. Here, we review studies reporting the concordance rates between cell-free deoxyribonucleic acid and trophectoderm biopsies, or whole blastocysts, in couples undergoing PGT. Noninvasive PGT results are promising for aneuploidy detection, with some early evidence of successful clinical application. Further research is required to explore its application for the detection of structural rearrangements and monogenic disorders.

Pre-Implantation Genetic Testing for Oculocutaneous Albinism Type 1 Using Karyomapping

Background: Oculocutaneous albinism type IA (OCA1) is the most severe form of albinism, an autosomal recessive inherited deficit of the pigment melanin causing distinctive alterations of skin, hair, and visual system. Pre-implantation genetic testing (PGT) is a substitution for prenatal diagnosis. Methods: This study accomplished SNP array with karyomapping for PGT of OCA1 and validated the results with PCR-based PGT. Results: One family with a risk of having OCA1 c.819+3insATATGCC and c.896G>A (p.R299H) offspring chose to go through karyomapping PGT. Novel PCR protocols employing fluorescent PCR and mini-sequencing were developed, tested, and applied. In the clinical PGT cycle, two blastocyst stage embryos were subjected to PGT. Karyotyping PGT results of OCA1 revealed both of the embryos to be normal. PCR analysis confirmed haplotyping results. However, copy number variation (CNV) analysis exhibited an additional chromosome 14 and segmental loss of 7q in embryo No. 1, i.e., 47, XY,+14,-7q, and an additional chromosome 22 in embryo No. 2, i.e., 47, XY,+22. Therefore, there was no appropriate embryo for transfer. The patient will return for the next PGT cycle. Conclusions: Karyomapping PGT for OCA1, including insertion c.819+3insATATGCC and point mutation c.896G>A (p.R299H), was performed alongside PCR techniques. Karyomapping gives benefits of CNV information to avoid the transfer of chromosomally unbalanced embryos.

Expanded clinical validation of Haploseek for comprehensive preimplantation genetic testing

PurposeWe previously developed Haploseek, a method for comprehensive preimplantation genetic testing (PGT). However, some key features were missing, and the method has not yet been systematically validated. MethodsWe extended Haploseek to incorporate DNA from embryo grandparents and to allow testing of variants on chromosome X or in regions where parents share common haplotypes. We then validated Haploseek on 151 embryo biopsies from 27 clinical PGT cases. We sequenced all biopsies to low coverage (0.2×), and performed single-nucleotide polymorphism (SNP) microarray genotyping on the embryos’ parents and siblings/grandparents. We used the extended Haploseek to predict chromosome copy-number variants (CNVs) and relevant variant-flanking haplotypes in each embryo. We validated haplotype predictions for each clinical sample against polymerase chain reaction (PCR)-based PGT case results, and CNV predictions against established commercial kits. ResultsFor each of the 151 embryo biopsies, all Haploseek-derived haplotypes and CNVs were concordant with clinical PGT results. The cases included 17 autosomal dominant, 5 autosomal recessive, and 3 X-linked monogenic disorders. In addition, we evaluated 1 Robertsonian and 2 reciprocal translocations, and 17 cases of chromosome copy-number counting were performed. ConclusionOur results demonstrate that Haploseek is clinically accurate and fit for all standard clinical PGT applications.

Consistency between chromosomal status analysis of biopsied human blastocyst trophectoderm cells and whole blastocyst cells.

PurposeThis study investigated the consistency between results of preimplantation genetic testing for aneuploidy performed on trophectoderm (TE) cells and remaining blastocyst cells.MethodsTE biopsy was performed on 29 surplus cryopreserved human blastocysts. Biopsy samples and remaining blastocysts were processed using the VeriSeq PGS kit, and chromosomal statuses were compared by next‐generation sequencing.ResultsDiscordance was observed in the chromosomal status of 11 out of 29 blastocysts between the biopsied TE and remaining blastocysts. Concordance was observed in 11 of 12 blastocysts classified as euploid by TE biopsy and in 7 of 17 blastocysts classified as aneuploid. There was 100% concordance (7/7) in cases diagnosed as aneuploid with no mosaicism by TE biopsy. However, discordance was observed in all 10 cases showing mosaicism or partial chromosomal abnormality.ConclusionChromosomal status analysis based on TE biopsy does not accurately reflect the chromosomal status of the whole blastocyst. The chromosomal status is usually the same between the TE and remaining blastocyst cells in cases diagnosed as euploid or aneuploid with no mosaicism. However, mosaic blastocysts and those with other types of structural rearrangements have a higher risk of inconsistency, warranting caution during embryo selection.

Application of single-sperm sequencing in a male with Marfan syndrome: a case report and a literature review

Marfan syndrome (MFS) is caused by a FBN1 mutation. Many organ systems are affected in patients with MFS, including the skeletal, ocular, cardiovascular and pulmonary systems. Cardiovascular manifestations are the main cause of mortality in patients with MFS. The mode of inheritance of MFS is autosomal dominant inheritance and the offspring are at great risk for the disease. Thus, the genetic testing for monogenic disease during preimplantation (PGT-M) is routinely advised for patients with MFS. PGT-M is a clinical genetic method to obtain normal embryos which are not affected by the monogenetic disorder. However, allele drop out (ADO) typically results in misdiagnosis during the PGT-M in the autosomal dominant disorder. Thus, a linkage analysis of polymorphic sites is used to identify ADO and improve the accuracy of PGT-M. However, when there are no family members affected, or the patients carry a de novo mutation, a linkage analysis cannot be performed to position the abnormal chromatid. Here, we performed single-sperm sequencing of preimplantation genetic testing in a male patient with MFS with a de novo mutation in FBN1. We constructed the chromosomal haplotype of the male patient by analysing information at the mutation site and at polymorphic sites. Next, the normal embryos were selected based on the results of high-throughput sequencing and haplotyping, and the one frozen embryo was transferred to the uterus. Finally, the preimplantation genetic testing results were confirmed by the prenatal genetic diagnosis during pregnancy, which showed that the foetus did not carry the pathogenic mutation. In conclusion, our research showed that single-sperm sequencing and haplotype analysis can be used in male patients with monogenetic disorders caused by de novo mutations to improve the accuracy of the preimplantation genetic diagnosis.

The Effectiveness of Next-Generation Sequencing-Based Preimplantation Genetic Testing for Balanced Translocation Couples

The purpose of this study was to evaluate the effectiveness of next-generation sequencing (NGS)-based preimplantation genetic testing (PGT) for balanced translocation carriers to identify normal/balanced blastocysts and to measure pregnancy outcomes following euploid embryo transfer. We enrolled 75 couples with a balanced translocation who underwent 83 PGT cycles (58 cycles for carriers with reciprocal translocations and 25 cycles for carriers with Robertsonian translocations) and 388 blastocysts were diagnosed. Moreover, we transferred single euploid blastocysts through frozen embryo transfer and calculated the biochemical pregnancy, clinical pregnancy, miscarriage, and ongoing pregnancy rates per embryo transfer cycle. Despite a mean maternal age of 29.8 years and mean of 4.34 embryos biopsied, there was a 32.8% chance of recording no chromosomally normal/balanced embryos for reciprocal translocation carriers. The proportion of normal/balanced embryos was significantly higher (44.1 vs. 27.8%) in Robertsonian translocation carriers than in reciprocal translocation carriers. Female carriers had a significantly lower (23.3 vs. 42.4%, 34.7 vs. 54.7%, respectively) percentage of normal/balanced embryos than male carriers, regardless of the translocation. After transfering single blastocysts, we obtained a 64.4% clinical pregnancy rate per transfer, and the clinical miscarriage rate was 5.7%. Amniocentesis results showed that all karyotypes of the fetuses were consistent with PGT results. The clinical outcomes are probably not influenced by the type of translocation, maternal age, and blastocyst morphology following the transfer of euploid blastocysts. Therefore, we conclude that NGS-based PGT is an efficient method for analyzing balanced translocation carriers, and aneuploidy screening had good clinical outcomes.

Successful four-factor preimplantation genetic testing: α- and β-thalassemia, human leukocyte antigen typing, and aneuploidy screening

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

The Incidence of Mosaicism for Individual Chromosome in Human Blastocysts Is Correlated With Chromosome Length.

Mosaicism, known as partial aneuploidies, mostly originates from mitotic errors during the post-zygotic stage; it consists of different cell lineages within a human embryo. The incidence of mosaicism has not been shown to correlate with maternal age, and its correlation with individual chromosome characteristics has not been well investigated. In this study, the results of preimplantation genetic testing for aneuploidy (PGT-A) derived from 4,036 blastocysts (930 IVF couples) were collected from 2015 to 2017. Via next-generation sequencing for comprehensive chromosome screening, embryo ploidy was identified as aneuploid, mosaic, and euploid. Total mosaicism was classified into two categories: “mosaic euploid/aneuploidy” (with mosaic aneuploidy between 20 and 80%) and “mosaic and aneuploidy” (a uniformly abnormal embryo superimposed with mosaic aneuploidies). Frequency of mosaicism was analyzed according to the function of chromosomal lengths, which divides involved chromosomes into three groups: group A (156–249 Mb), group B (102–145 Mb), and group C (51–90 Mb). The results show that the aneuploidy was more frequent in group C than in group A and group B (A: 23.7%, B: 35.1, 41.2%, p < 0.0001), while the mosaicism was more frequent in group A and group B than in group C [(Mosaic euploid/aneuploid) A: 14.6%, B: 12.4%, C: 9.9%, p < 0.0001; (mosaic and aneuploid) A: 21.3%, B: 22.9%, C: 18.9%, p < 0.0001; (Total mosaicism) A: 35.9%, B: 35.3%, C: 28.8%, p < 0.0001]. The significantly higher frequency of aneuploidy was on the shorter chromosome (< 90 Mb), and that of mosaicism was on the longer chromosomes (> 100 Mb). The length association did not reach significance in the patients with advanced age (≥ 36 years), and of the chromosome-specific mosaicism rate, the highest prevalence was on chromosome 14 (5.8%), 1 (5.7%), and 9 (5.6%). Although the length association was observed via group comparison, there may be affecting mechanisms other than chromosomes length. Eventually, twenty patients with mosaic embryo cryotransfers resulted in six live births. No significant correlation was observed between the transfer outcomes and chromosome length; however, the analysis was limited by small sample size.

Results of Preimplantation Genetic Testing for Aneuploidy (PGT-A) in a Cohort of 319 Embryos: Experience in a Fertility Clinic in Colombia.

ObjectiveTo analyze the results of the preimplantation genetic testing for aneuploidy at the Instituto de Fertilidad Humana - Inser Bogotá, Colombia, from 2016 to 2020.MethodsThis study is an observational, retrospective, and correlative analysis of biopsies from 319 embryos (from 54 patients) submitted to preimplantation genetic testing for aneuploidy by different molecular techniques.ResultsOf the 54 patients included in the study, 42 provided their own oocytes, and 12 used donated oocytes. The main indication to perform the preimplantation genetic testing was advanced maternal age. We obtained 319 embryos: Ninety-one (28.5%) euploid, 197 (61.8%) aneuploid and 31 (9.7%) with no detectable DNA. The highest rate of aneuploid embryos was found in patients over 40 years (72.7%), and the euploidy rate in patients under 35 years was 37.1%. After the transfer of euploid embryos, the rates for implantation, ongoing pregnancy, live birth, and miscarriage were 40%, 50%, 40.6%, and 0%, respectively. Older maternal age correlated with higher numbers of aneuploid embryos and lower numbers of both euploid and 5-day embryos.ConclusionsThere was a positive correlation between maternal age and aneuploidy rate. Complex chromosomal abnormalities were the most frequent aneuploidies, followed by mosaicism and double aneuploidies. The miscarriage rate after the transfer of euploid embryos was 0 %.

A whole-genome sequencing-based novel preimplantation genetic testing method for de novo mutations combined with chromosomal balanced translocations.

To explore a new preimplantation genetic testing (PGT) method for de novo mutations (DNMs) combined with chromosomal balanced translocations by whole-genome sequencing (WGS) using the MGISEQ-2000 sequencer. Two families, one with maternal Olmsted syndrome caused by DNM (c.1246C>T) in TRPV3 and a paternal Robertsonian translocation and one with paternal Marfan syndrome caused by DNM (c.4952_4955delAATG) in FBN1 and a maternal reciprocal translocation, underwent PGT for monogenetic disease (PGT-M), chromosomal aneuploidy, and structural rearrangement. WGS of embryos and family members were performed. Bioinformatics analysis based on gradient sequencing depth was performed, and parent-embryo haplotyping was conducted for DNM diagnosis. Sanger sequencing, karyotyping, and chromosomal microarray analysis were performed using an amniotic fluid sample to confirm the PGT results. After 1 PGT cycle, WGS of 2 embryos from the Olmsted syndrome family revealed euploid embryos without DNMs; after 2cycles, the 11 embryos from the Marfan syndrome family showed only 1 normal embryo without DNM, copy number variations (CNVs), or aneuploidy. Moreover, 1 blastocyst from the Marfan syndrome family was transferred back to the uterus; the amniocentesis test results were confirmed by PGT and a healthy infant was born. WGS based on parent-embryo haplotypes was an effective strategy for PGT of DNMs combined with a chromosomal balanced translocation. Our results indicate this is a reliable and effective diagnostic method that is useful for clinical application in PGT of patients with DNMs.

P0050PRE-IMPLANTATION GENETIC TESTING FOR MONOGENIC KIDNEY DISEASE: TWENTY-FIVE YEAR EXPERIENCE IN THE NETHERLANDS

Abstract Background and Aims Due to the fast-paced developments in human genetics, a genetic cause can now be identified for an increasing number of pediatric and adult-onset kidney diseases. A monogenic kidney disease (MKD) can impact prognostication and therapy decisions. It also provides patients with options regarding family planning such as preimplantation genetic testing (PGT), a reproductive technology that helps prospective parents prevent passing on a disease-causing mutation to their offspring. There are limited reports on PGT for MKD and most are focused on severe pediatric-onset diseases. The Dutch system of insurance reimbursement and nationwide regulation is unique, and it has allowed the Netherlands to be one of a handful of countries where couples have the option to choose PGT for MKD, including adult-onset forms of kidney disease. Here we provide the 25-year Dutch experience with PGT for MKD. Method We performed a retrospective cohort study of all couples counselled on PGT for MKD in the Maastricht University Medical Center+, the expert centre where all Dutch single cell genetic testing on embryos is performed, from January 1995 until June 2019. Results 99 couples were counseled for PGT, of which currently 15% is waiting for the genetic test to be validated. In the early years of PGT for MKD, referrals were incidental and only for couples at risk for offspring with paediatric-onset disease. From 2009 onwards, the number of referrals has steadily increased as has the number of couples referred for adult-onset MKD. Overall, the most frequent indications for referral for PGT were ADPKD (37%), X-linked Alport syndrome (25%) and ARPKD (9%). After counselling, 36% of n=99 couples started PGT treatment. In total n=80 cycles with oocyte retrieval were performed (median 2 cycles [range 1-4]). These cycles led to a median of n=10 embryos (range 2-31) suitable for biopsy and a median of n=3 embryos (range 1-14) being genetically unaffected. Seventy-five percent (n=) of couples achieved at least one live birth. Of n=99, 48% did not proceed with PGT, for various personal and technical reasons. Major reasons for opting out of PGT were that prospective parents did not want to wait for the lengthy PGT procedure (9%) or had a wish for natural conception with prenatal genetic testing (11%). Live birth rate was 40% (n=19) in the non-PGT group, with notably a similar duration to a live birth as the PGT group (2 years, range 0-5 years). Conclusion We provide the largest overview to date of the indications, uptake and results of PGT for MKD. Referrals for PGT, including adult-onset disease, have increased steadily over the past decade. PGT has favorable outcomes, with 75% of couples having at least one live birth, though this is likely due to our small sample size. Genetic and reproductive counseling, including information on PGT, should be provided to all patients and prospective parents from families with MKD to enable informed decision making.

Y/15 and Y/22 Derivative Chromosomes in Couples with Reproductive Failures: Algorithm of Preimplantation Genetic Testing and Specificity of Inheritance

The present study reports on the results of preimplantation genetic testing and IVF outcomes in four families, where one of the spouses is a carrier of Y/15 or Y/22 derivative chromosome: three cases of male carriers and one—of female carrier. In the cases of male carriers, we detected the predominant inheritance of the derivative chromosomes by the embryos and no abnormalities in sex chromosome segregation. In the case of female carrier, we did not observe either predominant inheritance of derivative chromosome by the embryos or effect of derivative chromosome on sex chromosome segregation. In all families, we revealed a high frequency of embryonic aneuploidies of chromosomes other than those involved in translocation. To exclude inheritance of the derivative chromosome by the offspring, preimplantation genetic testing should be recommended: fluorescent in situ hybridization (FISH), which allows identification of the Y-chromosome satellite DNA, followed by next generation sequencing (NGS) or microarray comparative genomic hybridization (aCGH) to detect other genomic and chromosomal abnormalities.

Eleven healthy live births: a result of simultaneous preimplantation genetic testing of α- and β-double thalassemia and aneuploidy screening.

To evaluate the efficacy of preimplantation genetic testing (PGT) for α- and β-double thalassemia combined with aneuploidy screening using next-generation sequencing (NGS). An NGS-based PGT protocol was performed between 2017 and 2018 for twelve couples, each of which carried both α- and β-thalassemia mutations. Trophectoderm biopsy samples underwent whole-genome amplification using multiple displacement amplification (MDA), followed by NGS for thalassemia detection and aneuploidy screening. A selection of several informative single nucleotide polymorphisms (SNPs) established haplotypes. Aneuploidy screening was performed only on unaffected noncarriers and carriers. Unaffected and euploid embryos were transferred into the uterus through frozen-thawed embryo transfer (FET). A total of 280 oocytes were retrieved following 18 ovum pick-up (OPU) cycles, with 182 normally fertilized and 112 cultured to become blastocysts. One hundredand seven (95.5%, 107/112) blastocysts received conclusive PGT results, showing 56 (52.3%, 56/107) were unaffected. Thirty-seven (66.1%, 37/56) of the unaffected were also identified as euploid. One family had no transferable embryos. Unaffected and euploid embryos were then transferred into the uterus of the other 11 couples resulting in 11 healthy live births. The clinical pregnancy rate was 61.1% (11/18) per OPU and 68.8% (11/16) per FET, with no miscarriage reported. Seven families accepted the prenatal diagnosis and received consistent results with the NGS-based PGT. This study indicated that NGS could realize the simultaneous PGT of double thalassemia and aneuploidy screening in a reliable and accurate manner. Moreover, it eliminated the need for multiple biopsies, alleviating the potential damages to the pre-implanted blastocysts.

Clinical utility of combined preimplantation genetic testing methods in couples at risk of passing on beta thalassemia/hemoglobin E disease: A retrospective review from a single center.

Thalassemia and hemoglobinopathy is a group of hereditary blood disorder with diverse clinical manifestation inherited by autosomal recessive manner. The Beta thalassemia/Hemoglobin E disease (HbE/βthal) causes a variable degree of hemolysis and the most severe form of HbE/βthal disease develop a lifelong transfusion-dependent anemia. Preimplantation genetic testing (PGT) is an established procedure of embryo genetic analysis to avoid the risk of passing on this particular condition from the carrier parents to their offspring. Preimplantation genetic testing for chromosomal aneuploidy (PGT-A) also facilitates the selection of embryos without chromosomal aberration resulting in the successful embryo implantation rate. Herein, we study the clinical outcome of using combined PGT-M and PGT-A in couples at risk of passing on HbE/βthal disease. The study was performed from January 2016 to December 2017. PGT-M was developed using short tandem repeat linkage analysis around the beta globin gene cluster and direct mutation testing using primer extension-based mini-sequencing. Thereafter, we recruited 15 couples at risk of passing on HbE/βthal disease who underwent a combined total of 22 IVF cycles. PGT was performed in 106 embryos with a 3.89% allele drop-out rate. Using combined PGT-M and PGT-A methods, 80% of women obtained satisfactory genetic testing results and were able to undergo embryo transfer within the first two cycles. The successful implantation rate was 64.29%. PGT accuracy was evaluated by prenatal and postnatal genetic confirmation and 100% had a genetic status consistent with PGT results. The overall clinical outcome of successful live birth for couples at risk of producing offspring with HbE/βthal was 53.33%. Conclusively, combined PGT-M and PGT-A is a useful technology to prevent HbE/βthal disease in the offspring of recessive carriers.

37. PREIMPLANTATION GENETIC TESTING: TO THE OPEN QUESTIONS ABOUT DATA INTERPRETATION OBTAINED BY MASSIVE PARALLEL SEQUENCING

Introduction the accuracy results of preimplantation genetic testing of embryos for aneuploidy (PGT-A) by massive parallel sequencing directly depends on the effectiveness of the quality control. The aim of this study was to analyze the external and intralaboratory system of the quality assessment to optimize protocols, algorithms of troubleshooting and selection of the most informative objective criteria for the interpretation of the PGT-A results. Materials and methods we analyzed the quality criteria more than 2000 human bioptates of the trophectoderm cells. PGT-A was performed using VeriSeq reagent kits. Sequencing was performed with the Illumina Miseq instrument. Sequencing data analysis was done by BlueFuse Multi Software. External quality assessment of PGT-A was carried out through participation in the program of interlaboratory comparisons of the consortium UK NEQAS. Results with the aim of obtaining optimal performance of the sequencing was carried out intralaboratory validation of methods of libraries preparation for sequencing. In 10% of cases, genomic profiles, which were difficult to interpret, were obtained with optimal performance criteria of whole genome amplification (WGA) and the quality of subsequent sequencing. One of the problems is the suspicion of segmental aneuploidies in a mosaic version. Recognition of false mosaicism helps, in particular, the absence of chromosome break points, the stereotypical pattern of chromosome copy number variations (CNV), which may be associated with difficulties in reading of GC-rich DNA regions. As a result of participation in the program of external quality assessment of a consortium UK NEQAS we found that partial DNA degradation in the sample makes it impossible to detect trisomies. Furthermore, studying the PGT-A statistical results of different laboratories, attention is drawn to a significant spread in the frequency of mosaicism detected by them. This may be primarily due to the complexity of interpreting so-called noisy CNV charts. When analyzing the sequencing results using BlueFuse Multi software, the researcher is able to estimate for each sample the total overall noise score, as well as the Region Confidence value for each chromosome. However, the low information content of these parameters to estimate the number of copies of a single chromosome often generates subjectivity in the decision of the expert. In this regard, the most important task is to minimize the technical factors that can lead to the appearance of intense artifacts in CNV-charts, which complicates their interpretation. We, together with the adjacent laboratory in Moscow, have developed a research proposal, the implementation of which will help to shed light on the technical issues of all embryo and PGT-A stages currently remain elusive. Conclusions the main reason for obtaining difficult to interpret genomic profiles is the low quality of the original DNA and as a consequence – false positive or false negative results of PGT-A. To prevent false-positive and false-negative results of PGT-A, a complex analysis of objective quality criteria of different stages of PGT analysis is necessary due to the lack of the information content of each individual factor in case of moderately pronounced DNA degradation in the sample.

65. SUCCESSFUL STRATEGY OF COMPREHENSIVE PRE-IMPLANTATION GENETIC TESTING FOR BETA-THALASSEMIA-HEMOGLOBIN E DISEASE AND CHROMOSOME BALANCE USING KARYOMAPPING

Introduction Thalassemia syndrome and hemoglobinopathy are the world commonest monogenic disease and cause health and economic burden worldwide. Pre-implantation genetic testing (PGT) is an alternative to traditional prenatal diagnosis (PND). However, the nature of a vast variety of mutations makes molecular genetic testing sophisticated and labor intensive. Modern haplotyping using SNP array (aSNP) and Karyomapping algorithm would omit molecular analysis development process and provide chromosome balance information at the same time. This study applied Karyomapping for PGT-M of beta-thalassemia-hemoglobin E (Hb E) disease in 2 clinical PGT cycles in comparison to PCR testing techniques. Material and methods: Two families at risk of having beta-thalassemia-Hb E disesase offspring decided to join the project following thoroughly counselling and inform consent was obtained. The patients underwent routine IVF procedures. Embryo biopsy was performed on Day-5 post-fertilization and the biopsied trophectoderm underwent whole genome amplification. SNP array with Karyomapping analysis was carried out for haplotyping as well as copy number variation (CNV). Multiplex PCR with mini-sequencing was performed alongside for confirmation standard molecular mutation analysis. Informative polymorphic marker was also included for contamination identification Results Thirteen and nine embryos with good morphology from families YW and CS were chosen for PGT, respectively. Karyomapping results of family YW (beta–thalassemia (c.17A>T)-Hb E (c.26G>A) disease) revealed four normal, two beta-thalassemia trait, one Hb E trait and six affected with beta-thalassemia-Hb E disease. Standard mutation analysis using multiplex fluorescent PCR and mini-sequencing confirmed haplotyping results in all embryos. In addition, Karyomapping demonstrated three embryos with chromosome unbalanced, i.e. 45,XY, -19 (affected), 47,XY, +16 (beta-thalassemia trait) and 47,XX, +21 (beta-thalassemia trait). Therefore, four normal (three male and one female) and one Hb E trait embryos were fulfilled for transfer. One normal male embryo was chosen for transfer and one normal male baby was delivered. Prenatal and postnatal DNA testing confirmed PGT results. Karyomapping results of family CS (beta–thalassemia (c.17A>T)-Hb E (c.26G>A) disease) revealed six Hb E trait and three affected with beta-thalassemia-Hb E disease. Standard mutation analysis using multiplex fluorescent PCR and mini-sequencing confirmed haplotyping results in all embryos. Additionally, Karyomapping demonstrated two embryos with chromosome unbalanced, i.e. 45,XY, -21 (affected) and 45,XY, -22 (Hb E trait). Therefore, five Hb E trait embryos were fulfilled for transfer. One Hb E trait embryo was chosen for transfer and one ongoing pregnancy was resulted. Polymorphic marker analysis revealed the absence of extraneous DNA contamination. Conclusions Two clinical PGT-M cycles using Karyomapping were performed for two families at risk of having beta–thalassemia (c.17A>T)-Hb E (c.26G>A) disease offspring. Additional standard multiplex PCR with mini-sequencing analysis confirmed haplotyping results of Karyomapping. However, aSNP provides the benefit of extra information of chromosome balance. This study demonstrated that kryomapping can omit the risk of transfer chromosomally unbalanced embryos, termination of abnormal chromosome pregnancy later and the birth of abnormal chromosome babies. Therefore, Karyomapping provides an accurate, quick, time saving for protocol development, universal PGT-M method for every monogenic disease, and also the advantage of CNV information which is common in pre-implantation embryos.