Results Of Preimplantation Genetic Testing Research Articles

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.

Next-generation sequencing analysis of embryos from mosaic patients undergoing in vitro fertilization and preimplantation genetic testing

To investigate the effects of parental mosaicism on their preimplantation embryos. Case series. An institute for reproductive and stem cell engineering. Sixty-eight mosaic couples. Assisted reproduction with preimplantation genetic testing (PGT). Karyotypes, embryo-related chromosomal abnormalities, and PGT results. A total of 209 embryos were obtained from 68 mosaic couples, and 153 (73.21%) of 209 of the total embryos were obtained from 55 mosaic couples with abnormal sex chromosome numbers. Of these 153 embryos, 2 (1.31%) had an abnormal copy number of X chromosome, 1 had mosaicism with 46,XN,+X(mosaic, 40%), 1 (0.65%) had an extra Y chromosome, 3 (1.96%) exhibited both X chromosomal and autosomal abnormalities, and 4 (2.61%) exhibited de novo X chromosome structural abnormalities. A total of 56 (26.79%) of 209 embryos were obtained from mosaic couples (n = 13) with abnormal autosomal structures. Notably, of these 56 embryos, 5 (8.93%) had a 16q21-q24.3 copy number abnormality related to the parental karyotype, with a fragile site at 16q22; 5 (7.14%) exhibited 46,XX,dup(8p23.1-8p11.21) and 46,XY,del(8p22-8p11.21), which were related to the parental karyotype; and 10 (17.86%) were de novo chromosome abnormalities. Our data demonstrate that the risk of embryo-related chromosome abnormalities in mosaic patients with abnormal sex chromosomes is very low. Therefore, PGT may not need to be recommended for mosaic patients with abnormal copy numbers of sex chromosomes, especially for patients with financial difficulties. By contrast, the mosaic patients with structural abnormalities of autosomes may have a relatively high risk of abnormal embryos with an unbalanced segment of the involved chromosomes. Thus, PGT is highly recommended for mosaic patients with autosomal structure abnormalities, especially those with a fragile site at 16q22.

Results of preimplantation genetic testing for aneuploidies as a basis for parental karyotypes correction

Introduction Chromosomal abnormalities are the most common genetic cause of infertility. An information about karyotypes of patients undergoing in vitro fertilization (IVF) is not always known. Therefore, IVF patients can be potential carriers of structural chromosomal rearrangements. We report three cases, in which results of preimplantation genetic testing for aneuploidies (PGT-A) allowed us to suspect the presence of parental reciprocal translocations. In each case, the parental reciprocal translocation was confirmed. Material & Methods PGT-A was performed by microarray comparative genomic hybridization (aCGH) (24sure, Illumina) or by next generation sequencing (NGS) (VeriSeq, Illumina). Indications for preimplantation testing were advanced maternal age (case 1 and 2) and paternal pericentric inversion of chromosome 10 (case 3). Parental karyotypes were not known before PGT-A in cases 1 and 2. In case 3 patients’ karyotypes was known before PGT-A and were 46,XX and 46,XY,inv(10)(p11.2;q22). The trophectoderm biopsy was performed at 5-6 day of embryo development. Analysis of parental karyotypes after obtaining PGT results was performed by standard cytogenetic study or by fluorescence in situ hybridization (FISH) on peripheral blood lymphocyte culture. The choice of method of parental karyotypes analysis was determined by size of the embryo chromosomal imbalance. Results PGT-A results showed a nonrandom pattern of segmental loses and gains in each case (table 1). These facts were the basis for parental karyotypes analysis. It was confirmed, that in each case one of the couple was the carrier of reciprocal translocation. In case 1 a paternal reciprocal translocation between chromosomes 11 and 22 was found – 46,XY,t(11;22)(q23.3;q13). In case 2 a maternal reciprocal translocation between chromosomes 10 and 12 was found – ish t(10;12)(D10S2290-, D12S158+; D10S2290+, D12S158-). In case 3 a maternal reciprocal translocation between chromosomes 2 and 12 was found - ish t(2;12)(D2S2147-,D12S399+; D2S2147+, D12S399-). Table 1 Case № Number of embryos Number of embryos with nonrandom pattern of segmental abnormalities Chromosomes involved in nonrandom segmental abnormalities Case 1 5 3 Chromosomes 11 and 22 Case 2 4 4 Chromosomes 10 and 12 Case 3 4 3 Chromosomes 2 and 12 Conclusions It is very importantto draw attention to pattern of segmental chromosomal abnormalities of embryos. 24-chromosome aneuploidy testing is not only an actual method for detection of embryo chromosomal imbalance, but in some cases also can help to correct parental karyotypes.

Karyomapping in Preimplantation Genetic Testing of Patients with Beta-thalassemia and Sickle Cell Anemia

Aim Preimplantation genetic testing (PGT) has been introduced to prevent the transmission of inherited diseases to the offspring. Karyomapping is a universal linkage-based test that has been recently introduced to PGT in the clinical practice. It utilizes single nucleotide polymorphism genotyping of individual parents along with a known disease reference from a family member for identification of the parental origin of chromosomes and the disease status of the embryos being tested. The objectives of this study were to first validate Karyomapping technique on previously known beta globin (HBB) test sample results and then establish as a sole PGT method for HBB mutation identification in the future. Materials and Methods First, Karyomapping protocol was validated on a total of 30 samples with HBB mutation identified by conventional PGT techniques. Then, Karyomapping was used on 31 samples as the sole technique to identify HBB mutation status. ResultsWe found a high concordance (97%) between the Karyomapping and conventional PGT results. For five PGT cycles for HBB mutations and direct Karyomapping PGT four unaffected and chromosomal normal embryos were transferred resulting two pregnancies. Conclusion: Karyomapping technique can reliably detect HBB mutation in clinical cases without a prior test development in the clinical practice and prevents the transfer of aneuploidy embryos.

A comparison of diagnostic results of preimplantation genetic testing for aneuploidy (PGT-A) from reference laboratories during a period of transition; trends and inherences for patient care

A comparison of diagnostic results of preimplantation genetic testing for aneuploidy (PGT-A) from reference laboratories during a period of transition; trends and inherences for patient care

The AUGMENTSM Treatment: Physician Reported Outcomes of the Initial Global Patient Experience

Background: Egg precursor cells can be readily isolated from the protective outer lining of the ovarian cortex. Studies have demonstrated that mitochondria isolated from these cells are of high quality. The AUGMENTSM treatment is a proprietary new technology based on previous clinical use demonstrating that the addition of mitochondria during in vitro fertilization (IVF) is safe, improves embryo quality, and increases the success of IVF. Subsequent published animal studies also confirmed the role of mitochondria in improved outcomes. This report represents the earliest global observations of the AUGMENTSM treatment in routine clinical practice from two distinct international centers. Methods and results: The AUGMENTSM treatment was initially used in a population of difficult-to-treat patients with a poor prognosis for success with standard IVF who were likely moving to donor egg as a next step. Each group reported marked improvements in pregnancy rates above the historic IVF success rate for these patients (e.g., 11- and 18- fold increase in ongoing clinical pregnancy rates in the UAE and Canada, respectively). In a 25 patient subset, retrieved eggs from each woman were allocated to two treatment groups; one group underwent the AUGMENTSM treatment at the time of ICSI while the other group underwent conventional ICSI. Embryos were selected for fresh-embryo transfer based on standard criteria including embryo morphology and the results of preimplantation genetic testing. Morphogenetic embryo selection and transfer from the AUGMENTSM treatment group was significantly higher, suggesting that improved embryo quality may have resulted in the improved pregnancy rates observed in these women. Conclusions: Based on these findings, the AUGMENTSM treatment may be a viable treatment option to address an unmet need for women with poor reproductive histories, history of poor egg quality, poor embryo development, previously failed IVF, and those seeking—despite repeated failures-to conceive genetically-related offspring.