Tomas Escudero

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).

Validation of microarray comparative genomic hybridization for comprehensive chromosome analysis of embryos

OBJECTIVE To validate and determine the best array-comparative genomic hybridization (aCGH; array-CGH) protocols for preimplantation genetic screening (PGS). DESIGN Embryos had one cell removed as a biopsy specimen and analyzed by one of two array-CGH protocols. Abnormal embryos were reanalyzed by fluorescence in situ hybridization (FISH). SETTING Reference laboratory. PATIENT(S) Patients donating embryos or undergoing PGS. INTERVENTION(S) Embryo biopsy, array-CGH, FISH reanalysis. MAIN OUTCOME MEASURE(S) Diagnosis, no result rate and error rate. RESULT(S) Method one produced 11.2% of embryos with no results and a 9.1% error rate compared with 3% and 1.9% for method two, respectively. Thereafter, only method two was used clinically. The aneuploidy rate for cleavage-stage embryos was 63.2%, significantly increasing with maternal age. The chromosomes most involved in aneuploidy were 16, 22, 21, and 15. We report the first live births after array-CGH combined with single blastomere biopsy. CONCLUSION(S) Array-CGH is proved to be highly robust (2.9% no results) and specific (1.9% error rate) when applied to rapid (24-hour) analysis of single cells biopsied from cleavage-stage embryos. This comprehensive chromosome analysis technique is the first to be validated by reanalyzing the same embryos with another technique (e.g., FISH). Unlike some alternative techniques for comprehensive chromosome screening, array-CGH does not require prior testing of parental DNA and thus advance planning and careful scheduling are unnecessary.

Outcome of preimplantation genetic diagnosis of translocations

OBJECTIVE To review 35 cases of preimplantation genetic diagnosis (PGD) of translocations with several methods, including telomeric probes. DESIGN Retrospective study. SETTING Clinical IVF laboratory. PATIENT(S) Thirty-five couples with one partner carrying a chromosomal translocation. INTERVENTION(S) PGD of translocation after polar-body or embryo biopsy. MAIN OUTCOME MEASURE(S) Pregnancy outcome. RESULT(S) Several trends were observed. First, PGD can achieve a statistically significant reduction in spontaneous abortion, from 95% to 13%. Second, the chances of achieving pregnancy are correlated with 50% or more of the embryos being chromosomally normal. Third, patients with robertsonian translocations produced fewer abnormal gametes and more pregnancies than did patients with reciprocal translocations. Fourth, a new fluorescence in situ hybridization protocol for PGD of translocations, which involves applying telomeric probes, has proved adequately reliable with a 6% average error rate. CONCLUSION(S) PGD of translocations achieves a statistically significant reduction in spontaneous abortion, both for polar-body and blastomere biopsy cases. Pregnancy outcome depended on the number of normal embryos available for transfer, with patients having <50% abnormal embryos achieving the most pregnancies. Because robertsonian translocations caused fewer abnormal embryos than reciprocal translocations, they also resulted in higher rates of implantation.

First clinical application of comparative genomic hybridization and polar body testing for preimplantation genetic diagnosis of aneuploidy.

Abstract Objective: To develop a preimplantation genetic diagnosis (PGD) protocol that allows any form of chromosome imbalance to be detected. Design: Case report employing a method based on whole-genome amplification and comparative genomic hybridization (CGH). Setting: Clinical IVF laboratory. Patient(s): A 40-year-old IVF patient. Intervention(s): Polar body and blastomere biopsy. Main Outcome Measure(s): Detection of aneuploidy. Result(s): Chromosome imbalance was detected in 9 of 10 polar bodies. A variety of chromosomes were aneuploid, but chromosomal size was found to be an important predisposing factor. In three cases, the resulting embryos could be tested using fluorescence in situ hybridization, and in each case the CGH diagnosis was confirmed. A single embryo could be recommended for transfer on the basis of the CGH data, but no pregnancy ensued. Conclusion(s): Evidence suggests that preferential transfer of chromosomally normal embryos can improve IVF outcomes. However, current PGD protocols do not allow analysis of every chromosome, and therefore a proportion of abnormal embryos remains undetected. We describe a method that allows every chromosome to be assessed in polar bodies and oocytes. The technique was accurate and allowed identification of aneuploid embryos that would have been diagnosed as normal by standard PGD techniques. As well as comprehensive cytogenetic analysis, this protocol permits simultaneous testing for multiple single-gene disorders.

Chromosome mosaicism in cleavage-stage human embryos: evidence of a maternal age effect

The present study evaluated mosaicism in a large series of cleavage-stage human embryos analysed by fluorescence in-situ hybridization. Only embryos with at least three cells analysed were included (n = 1235), of which 556 were mosaics. The most common types of mosaicism were chaotic (48%), diploid/polyploid (26%), and those caused by mitotic non-disjunction (25%). The number of abnormal cells per embryo ranged from 44% in diploid/polyploid to 84% in chaotic mosaics. Chromosome 16 was most commonly involved in mitotic non-disjunction mosaics. While overall mosaicism did not increase with maternal age, the average maternal age of the embryos that had mosaics caused by mitotic non-disjunction was significantly higher than that for normal or other mosaic embryos (P < 0.001). During the cleavage stage, the embryonic genome is not yet fully activated and consequently the mRNA and protein pools are still similar to those found in the oocyte. We therefore propose that the malfunctioning of the meiosis apparatus, which is similar to the mitotic one, may cause either meiotic errors or mitotic non-disjunction at cleavage-stage embryo development.

Chromosomal abnormalities in embryos derived from testicular sperm extraction

OBJECTIVE To compare the rate of chromosome abnormalities in embryos obtained from karyotypically normal patients with nonobstructive azoospermia undergoing testicular sperm extraction (TESE) to those from patients undergoing intracytoplasmic sperm injection (ICSI) with ejaculated sperm. DESIGN Retrospective analysis. SETTING IVF centers. PATIENT(S) Male partners had either nonobstructive zoospermia or oligospermia. INTERVENTION(S) Preimplantation genetic diagnosis. Chromosome enumeration was performed by fluorescence in situ hybridization (FISH). Embryos classified as abnormal were reanalyzed to study mosaicism. MAIN OUTCOME MEASURE(S) Chromosome abnormalities in embryos. RESULT(S) Embryos from ICSI cycles with ejaculated sperm (group 1) were 41.8% normal, 26.2% aneuploid, and 26.5% mosaic. In contrast, the embryos from ICSI cycles with TESE for nonobstructive azoospermia (group 2) were 22% normal, 17% aneuploid, and 53% mosaic. The difference in mosaicism rate between the two groups of embryos was highly significant. CONCLUSION(S) The present study results indicate a high incidence of mosaicism in embryos derived from TESE in men with a severe deficit in spermatogenesis. Sperm derived from TESE for nonobstructive azoospermia may have a higher rate of compromised or immature centrosome structures leading to mosaicism in the embryo.

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.

Predictive value of sperm fluorescence in situ hybridization analysis on the outcome of preimplantation genetic diagnosis for translocations

OBJECTIVE To determine whether the proportion of abnormal sperm is predictive of the proportion of abnormal embryos from couples in which the males are translocation carriers. DESIGN Controlled clinical study. SETTINGS Private in vitro fertilization (IVF) center. PATIENT(S) Eleven cases of reciprocal translocation male carriers. INTERVENTION(S) Blood sample and sperm sample collection from each male partner. Embryo biopsy of the embryos produced in each cycle. MAIN OUTCOME MEASURE(S) Fluorescence in situ hybridization on lymphocyte slides to characterize each translocation case, then fluorescent in situ hybridization (FISH) with specific probes for each of the sperm samples. Preimplantation genetic diagnosis of the translocations in the 11 cases. RESULTS A correlation was found between the percentage of abnormal gametes and the percentage of abnormal embryos, and a predictive equation is proposed for this relationship: A = -55 + (1.9 x B), where A is the percentage of abnormal embryos and B the percentage of abnormal sperm. CONCLUSION(S) The predictive value of the sperm analysis was established. Patients with 65% or less chromosomally abnormal sperm have a good chance at conceiving; patients with higher rates would need to produce 10 or more good quality embryos to have reasonable chances of conceiving.

Preimplantation genetic diagnosis significantly improves the pregnancy outcome of translocation carriers with a history of recurrent miscarriage and unsuccessful pregnancies

Preimplantation genetic diagnosis (PGD) for translocations has been shown to significantly reduce the risk of recurrent miscarriage, but because the majority of embryos produced are unbalanced, pregnancy rate is relatively low since 20% or more cycles have no normal or balanced embryos to transfer. The purpose of this study was to evaluate whether PGD could improve pregnancy outcome in translocation carriers with a history of two or more consecutive miscarriages and no live births. PGD for translocations was offered to translocation carriers with two or more previous miscarriages (average 3.5) and no live births (0/117 pregnancies) using a combination of distal and proximal probes to the breakpoints. After PGD, only 18.3% of embryos were normal or balanced. Only 5.3% of pregnancies were lost after PGD compared with 100% before PGD (P < 0.001). The cumulative pregnancy rate was 57.6% and the cumulative ongoing pregnancy rate was 54.5% in the short period of time of 1.24 IVF cycles, or 46.3% and 43.9% respectively per cycle. In conclusion, PGD significantly reduced losses and increased the number of viable pregnancies (P < 0.001). IVF plus PGD are a faster method of conceiving a live child than natural conception, at least for translocation carriers with recurrent miscarriages and no previous live births.