Silvia Fernández

Comprehensive embryo analysis of advanced maternal age–related aneuploidies and mosaicism by short comparative genomic hybridization

The short comparative genomic hybridization (short-CGH) method was used to perform a comprehensive cytogenetic study of isolated blastomeres from advanced maternal age embryos, discarded after fluorescent in situ hybridization (FISH) preimplantation genetic screening (PGS), detecting aneuploidies (38.5% of which corresponded to chromosomes not screened by 9-chromosome FISH), structural aberrations (31.8%), and mosaicism (77.3%). The short-CGH method was subsequently applied in one PGS, achieving a twin pregnancy.

Processing of semen can result in increased sperm DNA fragmentation

Processing of semen for assisted reproductive technologies (ART) entails a number of procedures that include semen liquefaction, removal of seminal plasma by centrifugation, incubation, and cryopreservation. The results of this study indicate that incubation of semen at room temperature and semen cryopreservation can result in increased levels of sperm DNA fragmentation.

A 24-chromosome FISH technique in preimplantation genetic diagnosis: validation of the method.

Abstract Embryo screening for aneuploidy (AS) is part of preimplantation genetic diagnostics (PGD) and is aimed at improving the efficiency of assisted reproduction. Currently, several technologies, including the well-established fluorescence in situ hybridization (FISH) technique, cover the screening of all chromosomes in a single cell. This study evaluates a novel 24-chromosome FISH technique protocol (FISH-24). A total of 337 embryos were analyzed using the traditional 9-chromosome FISH technique (FISH-9) while 251 embryos were evaluated using the new FISH-24 technique. Embryos deemed nontransferable on Day 3 were cultured in vitro to Day 5 of development, then fixed and reanalyzed according to the technique allocated to each treatment cycle (107 embryos analyzed by FISH-9 and 111 by FISH-24). The global error rate (discrepancy between Day 3 and Day 5 results for a single embryo) was 2.8% after FISH-9 and 3.6% after FISH-24, with a p value of 0.95. Thus, we have established and validated a 24-chromosome FISH-based single cell aneuploidy screening technique, showing that the error rate obtained for FISH-24 is independent of the number of chromosomes analyzed and equivalent to the error rate observed for FISH-9, as a useful tool for chromosome segregation studies and clinical use.

A study of meiotic segregation in a fertile human population following ovarian stimulation with recombinant FSH-LH

ObjectiveThe aim of the study is to investigate the meiotic segregation in fresh eggs from anonymous egg donors and to analyze the baseline levels of aneuploidy in this population.ResultsThe study includes the largest series of donor eggs so far studied: 203 eggs from donors aged between 20 and 31 years. No diagnosis was obtained in 10.8 % of cases (22/ 203). The biopsy of the first and second polar bodies was completed in a sequential manner on day 0 and day 1 of embryo development. Chromosomes 13, 16, 18, 21 and 22 are analyzed by means of the FISH test. The diagnosable fertilized eggs gave an aneuploidy rate of 19.1 % (31/162), with 83.8 % (26/31) of the errors produced during meiosis I, 12.9 % (4/31) produced during meiosis II, and 3.2 % (1/31) produced during both meiosis I and II. The premature division of sister chromatids is the main source of meiotic error during Meiosis I, resulting in the creation of oocyte aneuploidy.ConclusionsFISH analysis of the first and second polar body in donor oocytes gave an aneuploidy rate of 19.1 %. This study shows the majority of errors occur during Meiosis I.

Novel Double Factor PGT strategy analyzing blastocyst stage embryos in a single NGS procedure

In families at risk from monogenic diseases affected offspring, it is fundamental the development of a suitable Double Factor Preimplantation Genetic Testing (DF-PGT) method for both single-gene analysis and chromosome complement screening. Aneuploidy is not only a major issue in advanced-maternal-age patients and balanced translocation carriers, but also the aneuploidy rate is extremely high in patients undergoing in vitro fertilization (IVF), even in young donors. To adequate NGS technology to the DF-PGT strategy four different whole genome amplification systems (Sureplex, MALBAC, and two multiple displacement amplification systems-MDA) were tested using TruSight One panel on cell lines and blastocyst trophectoderm biopsies-TE. Embryo cytogenetic status was analyzed by Nexus software. Sureplex and MALBAC DNA products were considered not suitable for PGT diagnosis due to inconsistent and poor results on Trusight one (TSO) panel. Results obtained with both MDA based methods (GEH-MDA and RG-MDA) were appropriate for direct mutation detection by TSO NGS platform. Nevertheless, RG-MDA amplification products showed better coverage and lower ADO rates than GEH-MDA. The present work also demonstrates that the same TSO sequencing data is suitable not only for the direct mutation detection, but also for the indirect mutation detection by linkage analysis of informative SNPs. The present work also demonstrates that Nexus software is competent for the detection of CNV by using with TSO sequencing data from RG-MDA products, allowing for the whole cytogenetic characterization of the embryos. In conclusion, successfully development of an innovative and promising DF-PGT strategy using TSO-NGS technology in TE biopsies, performed in-house in a single laboratory experience, has been done in the present work. Additional studies should be performed before it could be used as a diagnostic alternative in order to validate this approach for the detection of chromosomal aneuploidies.