Mireia Sandalinas

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).

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.

Chromosome abnormalities in 1255 cleavage-stage human embryos

The relationship was examined between chromosome abnormalities in cleavage stage human embryos and maternal age, embryo morphology and development rate. Embryos that were classified as suboptimal for transfer from patients undergoing IVF treatment were disaggregated, and all or most of their cells were fixed for analysis by fluorescence in-situ hybridization. Chromosomes X, Y, 13, 18 and 21, and in some instances 16 were examined. A total of 731 non-viable embryos was analysed. An increase in chromosome abnormalities with decreasing embryo competence and increasing maternal age was shown. Compared with an earlier study, the major difference was that polyploidy (P<00.01) and aneuploidy were previously more common. After pooling results, it was found that aneuploidy increased with maternal age, from 3.1% in embryos from 20-34 years old patients to 17% in patients 40 years or older. Also, aneuploidy occurred more frequently in embryos with good morphology and development rate than in embryos developing poorly. In contrast, dysmorphic and slowly developing or arrested embryos had significantly more polyploidy and mosaicism than normally developing embryos. Clear associations between maternal age and aneuploidy, and between cleavage anomalies and mosaicism have been established in non-viable embryos. Arrested embryos were mostly polyploid. Moreover, polyploidy was found more frequently in embryos analysed on day 4, suggesting that developmentally compromised embryos became arrested in extended culture. A slightly higher aneuploidy rate in the earlier study may be attributed to differences in hormonal stimulation, which also resulted in different numbers of oocytes recruited and matured.

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.

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.

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.

Gamete segregation in female carriers of Robertsonian translocations

Eleven female carriers of either 45,XX,der(13;14) (q10;q10) or 45,XX,der(14;21)(q10;q10) underwent hormonal stimulation with the purpose of producing enough oocytes for in-vitro fertilization and preimplantation genetic diagnosis. Polar body biopsy was performed in those oocytes and FISH with painting probes was applied in their metaphase-like first polar body chromosomes. In this way, unbalanced, normal and balanced oocytes could be distinguished and segregation modes ascertained. der(14;21)(q10;q10) produced 42% unbalanced, 37% normal and 21% balanced oocytes (n = 86) while der(13;14)(q10;q10) generated 33% unbalanced, 51% normal and 16% balanced oocytes (n = 69). In both translocations the number of normal oocytes was significantly higher than the number of balanced oocytes. However, while the frequency of unbalanced events involving chromosome 13 and 14 was similar in der(13;14)(q10;q10), there were significantly more abnormalities involving chromosome 21 than 14 in the der(14;21) (q10;q10) cases. When comparing survival rates to term, trisomies from Robertsonian origin seem to survive more often than those originated by non-disjunction in non-translocation carriers. The meiotic segregation patterns found in female Robertsonian translocations are different from those described in male carriers, with higher rates of unbalanced gametes in females than in males.

Female gamete segregation in two carriers of translocations involving 2q and 14q

FISH, using a combination of whole‐chromosome painting and telomeric probes, was used to study the gamete segregation of two female carriers of translocations involving the same chromosome arms, 2q and 14q. Preimplantation genetic diagnosis of the first polar bodies of these oocytes permitted selecting normal embryos for replacement. Copyright

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.