Altered segregation pattern and numerical chromosome abnormalities interrelate in spermatozoa from Robertsonian translocation carriers.

The aim of this study was to assess whether there is a relationship between numerical chromosome abnormalities and certain segregation modes in spermatozoa from Robertsonian translocation carriers. A sequential fluorescence in-situ hybridization protocol based on two successive hybridization rounds was performed on sperm samples from one t(13;22) and ten t(13;14) carriers. Patient inclusion criteria included the presence of a positive interchromosomal effect (ICE). In the first round, numerical abnormalities for chromosomes 15/22, 18, 21, X and Y were analysed. In the second round, the segregation outcome of the rearranged chromosomes was evaluated in the numerically abnormal spermatozoa detected in the first round, as well as in randomly assessed spermatozoa. Aneuploid spermatozoa showed statistical differences in all segregation modes when compared with randomly assessed spermatozoa: alternate (50.7% versus 84.3%), adjacent (36.6% versus 14.6%) and 3:0 (10.2% versus 1%). Diploid/multiple disomic spermatozoa showed differences in alternate (3.7% versus 84.3%) and 3:0 (67.6% versus 1%). We concluded that in Robertsonian translocation carriers that exhibit ICE, numerically abnormal spermatozoa preferentially contain unbalanced segregation products. This might be explained by heterosynapsis acting as a rescue mechanism that would lead to aberrant recombination, which is a predisposing factor for non-disjunction events.

Substandard application of preimplantation genetic screening may interfere with its clinical success.

The intent of this study was to evaluate a recent randomized clinical trial evaluating the effect of preimplantation genetic screening (PGS) that reports a negative effect on pregnancy outcome. This article reviews appropriate PGS techniques and how they differ from the trial in question. A closer look at the clinical trial in question reveals significant lack of expertise in biopsy, cell fixation, genetic analysis, and patient selection. At most, this trial demonstrates that in inexperienced hands, PGS can be detrimental. No other conclusions concerning the effect of PGS on pregnancy results can be drawn from the trial.

PGD analysis for aneuploidy in a patient heterozygous for a polymorphism of chromosome 16 (16qh-).

OBJECTIVES: Detection and study of a polymorphism of chromosome 16qh in preimplantational embryos as well as in peripheral blood from the carrier. METHODS: A polymorphism of chromosome 16 (16qh-) was detected in PGD analysis for aneuploidy using a probe for the centromeric region of chromosome 16. The lack of pericentromeric heterochromatin in one of the chromosomes 16 could lead to misdiagnosis in PGD. PGD analysis using telomeric probes for this chromosome was performed to confirm the polymorphism as well as to avoid misdiagnosis in embryo blastomeres. FISH in lymphocyte metaphases was used to detect the carrier. RESULTS: Telomeric probes allowed detection of a polymorphism for the centromeric region of chromosome 16. FISH with centromeric and telomeric probes in lymphocyte metaphases of the parents showed that the patient was the carrier of the polymorphism. CONCLUSION: It is highly recommended that telomeric probes for chromosome 16 be used in preimplantation genetic diagnosis when a high frequency of monosomy for chromosome 16 is observed after regular aneuploidy analysis.

Increased rate of aneuploid embryos in young women with previous aneuploid conceptions.

OBJECTIVES: To determine whether a history of a previous aneuploid conception increases the rate of aneuploidy among women having preimplantation diagnosis (PGD). METHODS: Preimplantation embryos were tested for aneuploidy using FISH probes specific for chromosomes 13,15,16,17,18,21,22,X and Y. Using logistic regression to control for maternal age, we compared the rates of aneuploidy and other chromosome errors in 344 embryos from women having PGD because of a history of a previous aneuploid conception, 363 embryos from 42 women having PGD because of X-linked disorders and 1158 embryos from 135 women having PGD because of repeated in vitro fertilization failure. RESULTS: The frequency of aneuploidy differed significantly among patient groups for women younger than 35 (p = 0.003) but not for women older than 35. In women < 35, the rate of detected aneuploidy was 37.4% in the Aneuploid group, 20.9% in the X-linked group and 27.0% in the RIF group. (p = 0.0003 when the control groups are combined). The frequency of other chromosome abnormalities, as well as pregnancy and implantation rates, did not differ significantly among patient groups. CONCLUSIONS: This study suggests that a history of a trisomic pregnancy, whether or not it was a viable trisomy, is associated with an increased risk of another aneuploid conception at conception.

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.

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).

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 of numerical abnormalities for 13 chromosomes.

Several types of FISH protocols for PGD have been used to maximize results from a limited number of fluorochomes to study as many chromosomes as possible. The major purpose of the present study was to optimize the use of three sequential hybridizations to analyse up to 15 chromosome types in single cells. A secondary purpose was to study the frequency of aneuploidy of other chromosomes not yet extensively studied in preimplantation embryos. Patients underwent PGD of aneuploidy, and the biopsied cells were analysed with three sequential hybridizations, the first for chromosomes 13, 16, 18, 21 and 22, the second for X, Y, 15 and 17 and the third for 2, 3, 4 and 11. Overall, only 27% of embryos were normal. The chromosomes most involved in aneuploidy were, in order, chromosome 16, 15, 21, 22, 13, 18, 17, 3, 2, 4, 11, and gonosomes. Of the abnormal embryos, only 3% would have been missed without the third set of probes. This protocol allows the simultaneous analysis of up to 15 chromosomes although only 13 were analysed in this study. Results so far show that the chromosomes most involved in abnormalities are those already covered with the two first sets of probes.

Spectral karyotyping of fresh, non-inseminated oocytes.

The object of this study was to determine the mechanisms producing aneuploidy in female meiosis I by analysing the whole chromosome complement of human non-inseminated and unfertilized fresh oocytes. For this purpose, 131 fresh oocytes were obtained from 16 oocyte donors (24-48 years old). These oocytes were fixed immediately after retrieval and 47 good quality metaphases from 13 donors were analysed by spectral karyotyping to identify all 23 chromosome types. The data was divided into two maternal age groups, 24-34 (n = 31) and > or = 35 years (n = 16). More non-disjunction (13 and 25%), unbalanced predivision (10 and 44%, P < 0.01) and balanced predivision (6 and 62%, P < 0.001) events were found in the older group of oocytes. There was an increase in balanced predivision with decreasing chromosome size (P < 0.001). The present results are the first obtained in fresh oocytes where all chromosomes were specifically identified, and support previous theories that predivision of chromatids is a major factor causing aneuploidy. Previous reports with inseminated, non-fertilized oocytes fixed > or = 24 h after retrieval suffered from artefactual predivision of chromatids triggered by in-vitro ageing of oocytes.

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.