Esther Velilla

Improved implantation after preimplantation genetic diagnosis of aneuploidy

The objective of this study was to assess the improvement in implantation rates after preimplantation genetic diagnosis (PGD) of numerical abnormalities for the sole indication of advanced maternal age when compared with a control group. Each PGD patient was matched to a control patient according to several parameters prior to obtaining pregnancy results. The diagnosis was based on the analysis of chromosomes X, Y, 13, 15, 16, 18, 21 and 22 plus a ninth probe (1, 7, 14 or 17) on a single cell per embryo. The results were also analysed in relation to the previous number of IVF cycles and the number of dipronucleated zygotes obtained, when replacing presumptively chromosomally normal embryos on day 4 of development. It was found that women of advanced reproductive age (average age 40 years) had a higher implantation rate (18%) than their matched controls treated with standard IVF (11%) (P < 0.05). This increase was not observed in patients with two or more previous IVF cycles or patients with fewer than eight zygotes. Patients with eight or more 2PN zygotes and one or no previous cycles showed the greatest improvement in implantation rate, from 8.8% in controls to 19.2% in the PGD group (average age 40 years) (P < 0.025).

Differences in chromosome susceptibility to aneuploidy and survival to first trimester.

The purpose of this study was to find specific rates of aneuploidy in cleavage-stage embryos compared with first trimester data and to evaluate post-zygotic selection against aneuploidy. A total of 2058 embryos were analysed by flurorescence in-situ hybridization (FISH), and specific aneuploidy rates were obtained for 14 chromosomes. Data from morphologically abnormal embryos could be pooled with data from preimplantation genetic diagnosis (PGD) cycles because it was observed that they had similar rates of aneuploidy; thus, for the purpose of studying aneuploidy they could be, and were, pooled. Specific chromosome aneuploidy rates were not related to morphology or development of the embryos. The average maternal age of patients with aneuploid embryos was significantly higher than the overall analysed population. Monosomy appeared more commonly than trisomy. The chromosomes most frequently involved in aneuploidy were (in order) 22, 16, 21 and 15. When compared with first trimester pregnancy data, aneuploidies detected at cleavage stage seem to die in excess of 90% before reaching first trimester, with the exception of chromosome 16 and gonosomes (76% and 14% respectively). Differences in chromosome-specific aneuploidy rates at first trimester conceptions are probably produced by different chromosome-specific aneuploidy rates at cleavage stage and different survival rates to first trimester.

Blastomere fixation techniques and risk of misdiagnosis for preimplantation genetic diagnosis of aneuploidy

One of the most critical steps in preimplantation genetic diagnosis (PGD) studies is the fixation required to obtain good fluorescence in-situ hybridization (FISH) nuclear quality without losing any of the cells analysed. Different fixation techniques have been described. The aim of this study was to compare three fixation methods (1, acetic acid/methanol; 2, Tween 20; 3, Tween 20 and acetic acid/methanol) based on number of cells lost after fixation, average rate of informative cells, rate of signal overlaps and FISH errors. A total of 100, 106 and 114 blastomeres were fixed using techniques 1, 2 and 3 respectively. Technique 2 gave the poorest nuclear quality with higher cytoplasm, number of overlaps and FISH errors. Although technique 1 showed better nuclear quality in terms of greater nuclear diameter, fewer overlaps and FISH errors, it is difficult to perform correctly. However, technique 3 shows reasonably good nuclear quality and is both easier to learn and use for PGD studies than the others.

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.

Association between endometrial thickness in oocyte donation cycles and pregnancy success rates

Endometrial receptivity is a primary concern for embryo implantation success in fertility treatments. The present study was a retrospective analysis of 4070 cycles with donor oocytes and hormone-replacement therapy. Endometrial thickness was assessed once with transvaginal ultrasound. Patients were allowed to continue when endometrial thickness was ?5mm and had triple line morphology. Pregnancy rates, the number of gestational sacs and miscarriage rates were analysed in relation to endometrium status. Regression models were used to analyse associations, taking the day of embryo transfer into account. All patient parameters were homogeneous. Mean endometrial thickness was 7.24±1.66mm, the mean number of embryos transferred was 2.04±0.43, the pregnancy rate was 48.06% and sacs were present in 42.3% of cycles. There were no significant differences in pregnancy rates, number of gestational sacs and miscarriage rates for different endometrial thickness measurements. The present study is, to our knowledge, the largest study evaluating the role of endometrial thickness in oocyte donation cycles. Endometrial thickness >5mm is a reasonable parameter for determining treatment success, and once it is observed in a single ultrasonographic evaluation there is no need for subsequent monitoring and embryo transfer can be scheduled over the following 1-16 days, because the results are not compromised. This may lead to a significant reduction in time and cost in fertility clinics.

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.