Clinical application of a protocol based on universal next-generation sequencing for the diagnosis of beta-thalassaemia and sickle cell anaemia in preimplantation embryos.

RESEARCH QUESTION: Mutations of the beta-globin gene (HBB) cause beta-thalassaemia and sickle cell anaemia. These are the most common cause of severe inherited disease in humans. Traditional preimplantation genetic testing protocols for detecting HBB mutations frequently involve labour intensive, patient-specific test designs owing to the wide diversity of disease-associated HBB mutations. We, therefore, asked the question whether a universally applicable preimplantation genetic testing method can be developed to test for HBB gene mutations. DESIGN: A multiplex polymerase chain reaction protocol was designed, allowing simultaneous amplification of multiple overlapping DNA fragments encompassing the entire HBB gene sequence in addition to 17 characterized, closely linked single nucleotide polymorphisms (SNP). Amplicons were then analysed using a next-generation sequencing method, revealing mutations and SNP genotypes. The protocol was extensively validated, optimized and eventually clinically applied on whole-genome amplified DNA derived from embryos of three couples carrying different combinations of beta-thalassaemia mutations. RESULTS: The HBB mutation status and associated SNP haplotypes were successfully determined in all 21 embryos. Analysis of 141 heterozygous sites showed no instances of allele dropout and the test displayed 100% concordance compared with the results obtained from karyomapping. This suggests that the combination of trophectoderm biopsy and highly sensitive next-generation sequencing may provide superior accuracy than typically achieved using traditional preimplantation genetic testing methods. Importantly, no patient-specific test design or optimization was needed. CONCLUSIONS: It is hoped that protocols that deliver almost universally applicable low-cost tests, without compromising diagnostic accuracy, will improve patient access to preimplantation genetic testing, especially in less affluent parts of the world.

Analysis of implantation and ongoing pregnancy rates following the transfer of mosaic diploid-aneuploid blastocysts.

Preimplantation genetic testing for aneuploidy (PGT-A) is widely used in IVF and aims to improve outcomes by avoiding aneuploid embryo transfers. Chromosomal mosaicism is extremely common in early development and could affect the efficacy of PGT-A by causing incorrect embryo classification. Recent innovations have allowed accurate mosaicism detection in trophectoderm samples taken from blastocysts. However, there is little data concerning the impact of mosaicism on viability, and the optimal clinical pathway for such embryos is unclear. This study provides new information concerning the extent to which mosaic preimplantation embryos are capable of producing pregnancies and births. Archived trophectoderm biopsy specimens from transferred blastocysts were analyzed using next generation sequencing (NGS). Unlike other PGT-A methods, NGS accurately detects mosaicism in embryo biopsies. 44 mosaic blastocysts were identified. Their clinical outcomes were compared to 51 euploid blastocysts, derived from a well-matched, contemporary control group. Mosaic embryos were associated with outcomes that were significantly poorer than those of the control group: implantation 30.1 versus 55.8% (P = 0.038); miscarriage rate 55.6 versus 17.2% (P = 0.036); and ongoing pregnancy 15.4 versus 46.2% (P = 0.003). 61% of the mosaic errors affected whole chromosomes and 39% were segmental aneuploidies. Embryo viability is compromised by the presence of aneuploid cells. However, a minority of affected embryos can produce successful pregnancies. Hence, such embryos should not necessarily be excluded, but given a lower priority for transfer than those that are fully euploid. It is recommended that pregnancies established after mosaic embryo transfers be subjected to prenatal testing, with appropriate patient counselling.

Detection of Germline Mosaicism for Robertsonian Translocation 14;14: A Case Report.

Background: Chromosomal structural rearrangements can lead to fertility problems and recurrent miscarriages. The intricate interplay of genetics during human development can lead to subtle anomalies that may affect reproduction. Case Presentation: A 33-year-old woman sought fertility treatment after experiencing six miscarriages. Products of conception from the final pregnancy loss had been karyotyped, revealing a Robertsonian translocation (RT), involving chromosome 14. Fertility investigations showed low anti-Mullerian hormone (AMH) levels but otherwise normal female characteristics with normal sperm parameters of her husband were observed and both partners having a normal karyotype. Two embryos were transferred in an IVF cycle but neither resulted in a successful pregnancy. Subsequently, preimplantation genetic testing for aneuploidy (PGT-A) was applied to trophectoderm biopsy specimens from 4 embryos, which revealed abnormalities involving chromosome 14. Sperm aneuploidy testing failed to detect any increase in the incidence of aneuploidy affecting chromosome 14. Further embryos genetic testing indicated that all identified chromosome 14 abnormalities in the embryos had a maternal (oocyte) origin. Conclusion: This case underscores challenges in diagnosing and managing germline mosaicism in fertility. A maternal 14;14 Robertsonian translocation, undetected in the patient's blood but impacting oocytes, likely explains recurrent miscarriage and observed embryo aneuploidies. Genetic mosaicism in reproductive medicine highlights the necessity for advanced testing and personalized treatments. Data integration from various genetic analyses could enhance managing treatment expectations and improving fertility experiences.

Clinical application of sequencing-based methods for parallel preimplantation genetic testing for mitochondrial DNA disease and aneuploidy.

OBJECTIVE: To validate and apply a strategy permitting parallel preimplantation genetic testing (PGT) for mitochondrial DNA (mtDNA) disease and aneuploidy (PGT-A). DESIGN: Preclinical test validation and case reports. SETTING: Fertility centers. Diagnostics laboratory. PATIENTS: Four patients at risk of transmitting mtDNA disease caused by m.8993T>G (Patients A and B), m.10191T>G (Patient C), and m.3243A>G (Patient D). Patients A, B, and C had affected children. Patients A and D displayed somatic heteroplasmy for mtDNA mutations. INTERVENTIONS: Embryo biopsy, genetic testing, and uterine transfer of embryos predicted to be euploid and mutation-free. MAIN OUTCOME MEASURES: Test accuracy, treatment outcomes, and mutation segregation. RESULTS: Accuracy of mtDNA mutation quantification was confirmed. The test was compatible with PGT-A, and half of the embryos tested were shown to be aneuploid (16/33). Mutations were detected in approximately 40% of embryo biopsies from Patients A and D (10/24) but in none from Patients B and C (n = 29). Patients B and C had healthy children following PGT and natural conception, respectively. The m.8993T>G mutation displayed skewed segregation, whereas m.3243A>G mutation levels were relatively low and potentially impacted embryo development. CONCLUSIONS: Considering the high aneuploidy rate, strategies providing a combination of PGT for mtDNA disease and aneuploidy may be advantageous compared with approaches that consider only mtDNA. Heteroplasmic women had a higher incidence of affected embryos than those with undetectable somatic mutant mtDNA but were still able to produce mutation-free embryos. While not conclusive, the results are consistent with the existence of mutation-specific segregation mechanisms occurring during oogenesis and possibly embryogenesis.

The First ongoing Pregnancy Following Comprehensive Aneuploidy Assessment Using a Combined Blastocenetesis, Cell Free DNA and Trophectoderm Biopsy Strategy.

BACKGROUND: The exact origin of cell-free DNA found in spent culture media or blastocoel fluid is currently unknown but with the potential to become an improved source of DNA for chromosomal analysis than trophectoderm biopsy samples, it provides a superior representation of the fetal genetic status. However, the genetic material contained within the blastocoel cavity may be more reliable to assessment of embryo euploidy in a clinical context than trophectoderm of cell-free DNA. CASE PRESENTATION: This is the first UK case report where all three sources of DNA were analyzed in a clinical setting on 29 th January 2018 at the Centre for Reproductive and Genetic Health, London, leading to an ongoing clinical pregnancy. CONCLUSION: The experience from this case report suggests that removal of blastocoel fluid, sampling of spent culture media and trophectoderm biopsy can be carried out in parallel. Gathering genetic information from two to three independent samples of embryo DNA may provide enhanced diagnostic accuracy and may clarify cytogenetic status of mosaic embryos.

Detailed investigation into the cytogenetic constitution and pregnancy outcome of replacing mosaic blastocysts detected with the use of high-resolution next-generation sequencing.

OBJECTIVE: To determine the pregnancy outcome potential of mosaic embryos, detected by means of preimplantation genetic screening (PGS) with the use of next-generation sequencing (NGS). DESIGN: Retrospective study. SETTING: Genetics laboratories. PATIENT(S): PGS cycles during which either mosaic or euploid embryos were replaced. INTERVENTION(S): Blastocysts were biopsied and processed with the use of NGS, followed by frozen embryo transfer. Trophectoderm (TE) biopsies were classified as mosaic if they had 20%-80% abnormal cells. MAIN OUTCOME MEASURE(S): Implantation, miscarriage rates, and ongoing implantation rates (OIRs) were compared between euploid and types of mosaic blastocysts. RESULT(S): Complex mosaic embryos had a significantly lower OIR (10%) than aneuploidy mosaic (50%), double aneuploidy mosaic (45%), and segmental mosaic (41%). There was a tendency for mosaics with 40%-80% abnormal cells to have a lower OIR than those with <40% (22% vs. 56%). However, few embryos (n = 34) with a mosaic error in 40%-80% of the TE sample were replaced. There was no difference between monosomic and trisomic mosaics or between entire chromosome mosaicism or segmental mosaicism. Implantation rates were significantly higher (70% vs. 53%), miscarriage rates lower (10% vs. 25%), and OIRs higher (63% vs. 40%) after euploid embryo transfer than after mosaic embryo transfer. CONCLUSION(S): Forty-one percent of mosaic embryos produced an ongoing implantation. Complex mosaic blastocysts had a lower OIR than other mosaics. Mosaic monosomies performed as well as mosaic trisomies and mosaic segmental aneuploidies. The results suggest that embryos with >40% abnormal cells and those with multiple mosaic abnormalities (chaotic mosaics) are likely to have lower OIRs and should be given low transfer priority.